Bicyclic piperazine compounds

ABSTRACT

Bicyclic piperazine compounds of Formula I are provided, including stereoisomers, tautomers, and pharmaceutically acceptable salts thereof, useful for inhibiting Btk kinase, and for treating immune disorders such as inflammation mediated by Btk kinase. Methods of using compounds of Formula I for in vitro, in situ, and in vivo diagnosis, and treatment of such disorders in mammalian cells, or associated pathological conditions, are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This non-provisional application filed under 37 CFR §1.53(b), claims thebenefit under 35 USC §119(e) of U.S. Provisional Application Ser. No.61/555,396 filed on 3 Nov. 2011, which is incorporated by reference inentirety.

FIELD OF THE INVENTION

The invention relates generally to compounds for treating disordersmediated by Bruton's Tyrosine Kinase (Btk) including inflammation,immunological, and cancer, and more specifically to compounds whichinhibit Btk activity. The invention also relates to methods of using thecompounds for in vitro, in situ, and in vivo diagnosis or treatment ofmammalian cells, or associated pathological conditions.

BACKGROUND OF THE INVENTION

Protein kinases, the largest family of human enzymes, encompass wellover 500 proteins. Bruton's Tyrosine Kinase (Btk) is a member of the Tecfamily of tyrosine kinases, and is a regulator of early B-celldevelopment as well as mature B-cell activation, signaling, andsurvival.

B-cell signaling through the B-cell receptor (BCR) can lead to a widerange of biological outputs, which in turn depend on the developmentalstage of the B-cell. The magnitude and duration of BCR signals must beprecisely regulated. Aberrant BCR-mediated signaling can causedisregulated B-cell activation and/or the formation of pathogenicauto-antibodies leading to multiple autoimmune and/or inflammatorydiseases. Mutation of Btk in humans results in X-linkedagammaglobulinaemia (XLA). This disease is associated with the impairedmaturation of B-cells, diminished immunoglobulin production, compromisedT-cell-independent immune responses and marked attenuation of thesustained calcium sign upon BCR stimulation. Evidence for the role ofBtk in allergic disorders and/or autoimmune disease and/or inflammatorydisease has been established in Btk-deficient mouse models. For example,in standard murine preclinical models of systemic lupus erythematosus(SLE), Btk deficiency has been shown to result in a marked ameliorationof disease progression. Moreover, Btk deficient mice can also beresistant to developing collagen-induced arthritis and can be lesssusceptible to Staphylococcus-induced arthritis. A large body ofevidence supports the role of B-cells and the humoral immune system inthe pathogenesis of autoimmune and/or inflammatory diseases.Protein-based therapeutics (such as Rituxan) developed to depleteB-cells, represent an approach to the treatment of a number ofautoimmune and/or inflammatory diseases. Because of Btk's role in B-cellactivation, inhibitors of Btk can be useful as inhibitors of B-cellmediated pathogenic activity (such as autoantibody production). Btk isalso expressed in osteoclasts, mast cells and monocytes and has beenshown to be important for the function of these cells. For example, Btkdeficiency in mice is associated with impaired IgE-mediated mast cellactivation (marked diminution of TNF-alpha and other inflammatorycytokine release), and Btk deficiency in humans is associated withgreatly reduced TNF-alpha production by activated monocytes.

Thus, inhibition of Btk activity can be useful for the treatment ofallergic disorders and/or autoimmune and/or inflammatory diseases suchas: SLE, rheumatoid arthritis, multiple vasculitides, idiopathicthrombocytopenic purpura (ITP), myasthenia gravis, allergic rhinitis,and asthma (Di Paolo et al (2011) Nature Chem. Biol. 7(1):41-50; Liu etal (2011) Jour. of Pharm. and Exper. Ther. 338(1):154-163). In addition,Btk has been reported to play a role in apoptosis; thus, inhibition ofBtk activity can be useful for cancer, as well as the treatment ofB-cell lymphoma, leukemia, and other hematological malignancies.Moreover, given the role of Btk in osteoclast function, the inhibitionof Btk activity can be useful for the treatment of bone disorders suchas osteoporosis. Specific Btk inhibitors have been reported (Liu (2011)Drug Metab. and Disposition 39(10):1840-1849; U.S. Pat. No. 7,884,108,WO 2010/056875; U.S. Pat. No. 7,405,295; U.S. Pat. No. 7,393,848; WO2006/053121; U.S. Pat. No. 7,947,835; US 2008/0139557; U.S. Pat. No.7,838,523; US 2008/0125417; US 2011/0118233; PCT/US2011/050034“PYRIDINONES/PYRAZINONES, METHOD OF MAKING, AND METHOD OF USE THEREOF”,filed 31 Aug. 2011; PCT/US2011/050013 “PYRIDAZINONES, METHOD OF MAKING,AND METHOD OF USE THEREOF”, filed 31 Aug. 2011; U.S. Ser. No. 13/102,720“PYRIDONE AND AZA-PYRIDONE COMPOUNDS AND METHODS OF USE”, filed 6 May2011).

SUMMARY OF THE INVENTION

The invention relates generally to Formula I, bicyclic piperazinecompounds with Bruton's Tyrosine Kinase (Btk) modulating activity.

Formula I compounds have the structure:

including stereoisomers, tautomers, or pharmaceutically acceptable saltsthereof. The various substituents are defined herein below.

One aspect of the invention is a pharmaceutical composition comprised ofa Formula I compound and a pharmaceutically acceptable carrier, glidant,diluent, or excipient. The pharmaceutical composition may furthercomprise a second therapeutic agent.

Another aspect of the invention is a process for making a pharmaceuticalcomposition which comprises combining a Formula I compound with apharmaceutically acceptable carrier.

The invention includes a method of treating a disease or disorder whichmethod comprises administering a therapeutically effective amount of aFormula I compound to a patient with a disease or disorder selected fromimmune disorders, cancer, cardiovascular disease, viral infection,inflammation, metabolism/endocrine function disorders and neurologicaldisorders, and mediated by Bruton's tyrosine kinase.

The invention includes a kit for treating a condition mediated byBruton's tyrosine kinase, comprising: a) a first pharmaceuticalcomposition comprising a Formula I compound; and b) instructions foruse.

The invention includes a Formula I compound for use as a medicament, andfor use in treating a disease or disorder selected from immunedisorders, cancer, cardiovascular disease, viral infection,inflammation, metabolism/endocrine function disorders and neurologicaldisorders, and mediated by Bruton's tyrosine kinase.

The invention includes use of a Formula I compound in the manufacture ofa medicament for the treatment of immune disorders, cancer,cardiovascular disease, viral infection, inflammation,metabolism/endocrine function disorders and neurological disorders, andwhere the medicament mediates Bruton's tyrosine kinase.

The invention includes methods of making a Formula I compound.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a shows the preparation of2-(5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-a]pyridin-6-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one101 starting with 6-Chloro-8-bromoimidazo[1,2-a]pyridine 101a.

FIG. 1 b shows the preparation of4-Fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylacetate 101l starting with3,4,6,7,8,9-Hexahydropyrazino[1,2-a]indol-1(2H)-one 101g and2,6-Dibromo-4-fluorobenzyl Acetate 101j

FIG. 2 shows the preparation of2-(5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazine-1-yl)pyridine-2-ylamino)imidazo[1,2-a]pyridin-6-yl)phenyl)-3,4,6,7,8,9-hexahydro-pyrazino[1,2-a]indol-1(2H)-one102 starting with tert-Butyl4-(6-(6-chloroimidazo[1,2-a]pyridin-8-ylamino)pyridine-3-yl)piperazine-1-carboxylate102a

FIG. 3 shows the preparation of5-[5-Fluoro-2-(hydroxymethyl)-3-{(8-(5-(4-methylpiperazin-1-yl)pyridine-2-ylamino)imidazo[1,2-a]pyridin-6-yl)}phenyl]-8-thia-5-azatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7)-dien-6-one103 starting with2-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-4-fluoro-6-(1-oxo-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-2(1H)-yl)benzylAcetate 103g

FIG. 4 shows the preparation of2-(5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one104 starting with 8-Bromo-6-chloroimidazo[1,2-b]pyridazine 104a

FIG. 5 shows the preparation of2-(5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)phenyl)-3,4,6,7,8,9-hexahydro-pyrazino[1,2-a]indol-1(2H)-one105 starting with6-Chloro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)imidazo[1,2-b]pyridazin-8-amine105a

FIG. 6 shows the preparation of2-(5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-a]pyrazin-6-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one106 starting with tert-Butyl4-(6-(6-Bromoimidazo[1,2-a]pyrazin-8-ylamino)pyridin-3-yl)piperazine-1-carboxylate106a

FIG. 7 shows the preparation of2-(5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-a]pyrazin-6-yl)phenyl)-3,4,6,7,8,9-hexahydro-pyrazino[1,2-a]indol-1(2H)-one107 starting with2-(5-Fluoro-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-imidazo[1,2-a]pyrazin-6-yl)-2-(2-oxopropyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one107a

FIG. 8 shows the preparation of2-(5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-[1,2,4]triazolo[1,5-a]pyridin-6-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one108 starting with(E)-N′-(3-Bromo-5-chloropyridin-2-yl)-N,N-dimethylformimidamide 108a

FIG. 9 shows the preparation of2-(5-Fluoro-2-(hydroxymethyl)-3-(3-methyl-7-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-3H-benzo[d]imidazol-5-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one109 starting with 2-Bromo-4-chloro-6-nitrobenzenamine 109a

FIG. 10 shows the preparation of10-[5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-9-one110 starting with2-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-4-fluoro-6-(9-oxo-4,4-dimethyl-1,10diazatricyclo[6.4.0.0^(2,6)]-dodeca-2(6),7-dien-10-yl)benzylAcetate 110g and6-chloro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)imidazo[1,2-a]pyridin-8-amine

FIG. 11 shows the preparation of5-[5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)phenyl]-8-thia-5-azatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7)-dien-6-one111 starting with(4-fluoro-2-{6-oxo-8-thia-5-azatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methylacetate 103g and6-chloro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)imidazo[1,2-b]pyridazin-8-amine.

FIG. 12 shows the preparation of5-[5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)phenyl]-8-thia-4,5-diazatricyclo-[7.4.0.0^(2,7)]trideca-1(9),2(7),3-trien-6-one112 starting with(4-fluoro-2-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7),3-trine-5-yl}-6-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methylacetate and6-chloro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)imidazo[1,2-b]pyridazin-8-amine

FIG. 13 shows the preparation of10-[5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridine-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0^(2,6)]dodeca-1(8),2(6)-dien-9-one113 starting with(2-{4,4-Dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0^(2,6)]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methylAcetate 113j and6-chloro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)imidazo[1,2-a]pyridin-8-amine.

FIG. 14 shows the preparation of2-(3-(Hydroxymethyl)-4-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)pyridin-2-yl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one114 starting with(2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)methylacetate 114e and6-chloro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridine-2-yl)imidazo[1,2-b]pyridazin-8-amine.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents which may be included within the scope ofthe present invention as defined by the claims. One skilled in the artwill recognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention. The present invention is in no way limited to the methods andmaterials described. In the event that one or more of the incorporatedliterature, patents, and similar materials differs from or contradictsthis application, including but not limited to defined terms, termusage, described techniques, or the like, this application controls.Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. The nomenclature used in this Application is based on IUPACsystematic nomenclature, unless indicated otherwise.

DEFINITIONS

when indicating the number of substituents, the term “one or more”refers to the range from one substituent to the highest possible numberof substitution, i.e. replacement of one hydrogen up to replacement ofall hydrogens by substituents. The term “substituent” denotes an atom ora group of atoms replacing a hydrogen atom on the parent molecule. Theterm “substituted” denotes that a specified group bears one or moresubstituents. Where any group may carry multiple substituents and avariety of possible substituents is provided, the substituents areindependently selected and need not to be the same. The term“unsubstituted” means that the specified group bears no substituents.The term “optionally substituted” means that the specified group isunsubstituted or substituted by one or more substituents, independentlychosen from the group of possible substituents. When indicating thenumber of substituents, the term “one or more” means from onesubstituent to the highest possible number of substitution, i.e.replacement of one hydrogen up to replacement of all hydrogens bysubstituents.

The term “alkyl” as used herein refers to a saturated linear orbranched-chain monovalent hydrocarbon radical of one to twelve carbonatoms (C₁-C₁₂), wherein the alkyl radical may be optionally substitutedindependently with one or more substituents described below. In anotherembodiment, an alkyl radical is one to eight carbon atoms (C₁-C₈), orone to six carbon atoms (C₁-C₆). Examples of alkyl groups include, butare not limited to, methyl (Me, —CH₃), ethyl (Et, —CH₂CH₃), 1-propyl(n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr, i-propyl, —CH(CH₃)₂),1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃), 2-methyl-1-propyl (i-Bu,i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl, —CH(CH₃)CH₂CH₃),2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl (n-pentyl,—CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃, 1-heptyl, 1-octyl, and the like.

The term “alkylene” as used herein refers to a saturated linear orbranched-chain divalent hydrocarbon radical of one to twelve carbonatoms (C₁-C₁₂), wherein the alkylene radical may be optionallysubstituted independently with one or more substituents described below.In another embodiment, an alkylene radical is one to eight carbon atoms(C₁-C₈), or one to six carbon atoms (C₁-C₆). Examples of alkylene groupsinclude, but are not limited to, methylene (—CH₂—), ethylene (—CH₂CH₂—),propylene (—CH₂CH₂CH₂—), and the like.

The term “alkenyl” refers to linear or branched-chain monovalenthydrocarbon radical of two to eight carbon atoms (C₂-C₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp² double bond,wherein the alkenyl radical may be optionally substituted independentlywith one or more substituents described herein, and includes radicalshaving “cis” and “trans” orientations, or alternatively, “E” and “Z”orientations. Examples include, but are not limited to, ethylenyl orvinyl (—CH═CH₂), allyl (—CH₂CH═CH₂), and the like.

The term “alkenylene” refers to linear or branched-chain divalenthydrocarbon radical of two to eight carbon atoms (C₂-C₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp² double bond,wherein the alkenylene radical may be optionally substituted substitutedindependently with one or more substituents described herein, andincludes radicals having “cis” and “trans” orientations, oralternatively, “E” and “Z” orientations. Examples include, but are notlimited to, ethylenylene or vinylene (—CH═CH—), allyl (—CH₂CH═CH—), andthe like.

The term “alkynyl” refers to a linear or branched monovalent hydrocarbonradical of two to eight carbon atoms (C₂-C₈) with at least one site ofunsaturation, i.e., a carbon-carbon, sp triple bond, wherein the alkynylradical may be optionally substituted independently with one or moresubstituents described herein. Examples include, but are not limited to,ethynyl (—C≡CH), propynyl (propargyl, —CH₂C≡CH), and the like.

The term “alkynylene” refers to a linear or branched divalenthydrocarbon radical of two to eight carbon atoms (C₂-C₈) with at leastone site of unsaturation, i.e., a carbon-carbon, sp triple bond, whereinthe alkynylene radical may be optionally substituted independently withone or more substituents described herein. Examples include, but are notlimited to, ethynylene (—C≡C—), propynylene (propargylene, —CH₂C≡C—),and the like.

The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring” and“cycloalkyl” refer to a monovalent non-aromatic, saturated or partiallyunsaturated ring having 3 to 12 carbon atoms (C₃-C₁₂) as a monocyclicring or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycleshaving 7 to 12 atoms can be arranged, for example, as a bicyclo [4,5],[5,5], [5,6] or [6,6] system, and bicyclic carbocycles having 9 or 10ring atoms can be arranged as a bicyclo [5,6] or [6,6] system, or asbridged systems such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane andbicyclo[3.2.2]nonane. Spiro moieties are also included within the scopeof this definition. Examples of monocyclic carbocycles include, but arenot limited to, cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,cycloundecyl, cyclododecyl, and the like. Carbocyclyl groups areoptionally substituted independently with one or more substituentsdescribed herein.

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbonatoms (C₆-C₂₀) derived by the removal of one hydrogen atom from a singlecarbon atom of a parent aromatic ring system. Some aryl groups arerepresented in the exemplary structures as “Ar”. Aryl includes bicyclicradicals comprising an aromatic ring fused to a saturated, partiallyunsaturated ring, or aromatic carbocyclic ring. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene (phenyl),substituted benzenes, naphthalene, anthracene, biphenyl, indenyl,indanyl, 1,2-dihydronaphthalene, 1,2,3,4-tetrahydronaphthyl, and thelike. Aryl groups are optionally substituted independently with one ormore substituents described herein.

“Arylene” means a divalent aromatic hydrocarbon radical of 6-20 carbonatoms (C₆-C₂₀) derived by the removal of two hydrogen atom from a twocarbon atoms of a parent aromatic ring system. Some arylene groups arerepresented in the exemplary structures as “Ar”. Arylene includesbicyclic radicals comprising an aromatic ring fused to a saturated,partially unsaturated ring, or aromatic carbocyclic ring. Typicalarylene groups include, but are not limited to, radicals derived frombenzene (phenylene), substituted benzenes, naphthalene, anthracene,biphenylene, indenylene, indanylene, 1,2-dihydronaphthalene,1,2,3,4-tetrahydronaphthyl, and the like. Arylene groups are optionallysubstituted with one or more substituents described herein.

The terms “heterocycle,” “heterocyclyl” and “heterocyclic ring” are usedinterchangeably herein and refer to a saturated or a partiallyunsaturated (i.e., having one or more double and/or triple bonds withinthe ring) carbocyclic radical of 3 to about 20 ring atoms in which atleast one ring atom is a heteroatom selected from nitrogen, oxygen,phosphorus and sulfur, the remaining ring atoms being C, where one ormore ring atoms is optionally substituted independently with one or moresubstituents described below. A heterocycle may be a monocycle having 3to 7 ring members (2 to 6 carbon atoms and 1 to 4 heteroatoms selectedfrom N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9carbon atoms and 1 to 6 heteroatoms selected from N, O, P, and S), forexample: a bicyclo[4,5], [5,5], [5,6], or [6,6] system. Heterocycles aredescribed in Paquette, Leo A.; “Principles of Modern HeterocyclicChemistry” (W.A. Benjamin, New York, 1968), particularly Chapters 1, 3,4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series ofMonographs” (John Wiley & Sons, New York, 1950 to present), inparticular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960)82:5566. “Heterocyclyl” also includes radicals where heterocycleradicals are fused with a saturated, partially unsaturated ring, oraromatic carbocyclic or heterocyclic ring. Examples of heterocyclicrings include, but are not limited to, morpholin-4-yl, piperidin-1-yl,piperazinyl, piperazin-4-yl-2-one, piperazin-4-yl-3-one,pyrrolidin-1-yl, thiomorpholin-4-yl, S-dioxothiomorpholin-4-yl,azocan-1-yl, azetidin-1-yl, octahydropyrido[1,2-a]pyrazin-2-yl,[1,4]diazepan-1-yl, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl,tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino,thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl,thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl,thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl,4H-pyranyl, dioxanyl, 1,3-dioxolanyl, pyrazolinyl, dithianyl,dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,pyrazolidinylimidazolinyl, imidazolidinyl, 3-azabicyco[3.1.0]hexanyl,3-azabicyclo[4.1.0]heptanyl, azabicyclo[2.2.2]hexanyl, 3H-indolylquinolizinyl and N-pyridyl ureas. Spiro moieties are also includedwithin the scope of this definition. Examples of a heterocyclic groupwherein 2 ring atoms are substituted with oxo (═O) moieties arepyrimidinonyl and 1,1-dioxo-thiomorpholinyl. The heterocycle groupsherein are optionally substituted independently with one or moresubstituents described herein.

The term “heteroaryl” refers to a monovalent aromatic radical of 5-, 6-,or 7-membered rings, and includes fused ring systems (at least one ofwhich is aromatic) of 5-20 atoms, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Examples ofheteroaryl groups are pyridinyl (including, for example,2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl(including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl,oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl,isoquinolinyl, tetrahydroisoquinolinyl, indolyl, benzimidazolyl,benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl,pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl,triazolyl, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl,benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl,quinoxalinyl, naphthyridinyl, and furopyridinyl. Heteroaryl groups areoptionally substituted independently with one or more substituentsdescribed herein.

The heterocycle or heteroaryl groups may be carbon (carbon-linked), ornitrogen (nitrogen-linked) bonded where such is possible. By way ofexample and not limitation, carbon bonded heterocycles or heteroarylsare bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5,or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline.

By way of example and not limitation, nitrogen bonded heterocycles orheteroaryls are bonded at position 1 of an aziridine, azetidine,pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline,1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of amorpholine, and position 9 of a carbazole, or β-carboline.

The terms “treat” and “treatment” refer to therapeutic treatment,wherein the object is to slow down (lessen) an undesired physiologicalchange or disorder, such as the development or spread of arthritis orcancer. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, alleviation of symptoms,diminishment of extent of disease, stabilized (i.e., not worsening)state of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. Those in need of treatment include those with the conditionor disorder.

The phrase “therapeutically effective amount” means an amount of acompound of the present invention that (i) treats the particulardisease, condition, or disorder, (ii) attenuates, ameliorates, oreliminates one or more symptoms of the particular disease, condition, ordisorder, or (iii) prevents or delays the onset of one or more symptomsof the particular disease, condition, or disorder described herein. Inthe case of cancer, the therapeutically effective amount of the drug mayreduce the number of cancer cells; reduce the tumor size; inhibit (i.e.,slow to some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy can be measured, for example, by assessing the time to diseaseprogression (TTP) and/or determining the response rate (RR).

“Inflammatory disorder” as used herein can refer to any disease,disorder, or syndrome in which an excessive or unregulated inflammatoryresponse leads to excessive inflammatory symptoms, host tissue damage,or loss of tissue function. “Inflammatory disorder” also refers to apathological state mediated by influx of leukocytes and/or neutrophilchemotaxis.

“Inflammation” as used herein refers to a localized, protective responseelicited by injury or destruction of tissues, which serves to destroy,dilute, or wall off (sequester) both the injurious agent and the injuredtissue. Inflammation is notably associated with influx of leukocytesand/or neutrophil chemotaxis. Inflammation can result from infectionwith pathogenic organisms and viruses and from noninfectious means suchas trauma or reperfusion following myocardial infarction or stroke,immune response to foreign antigen, and autoimmune responses.Accordingly, inflammatory disorders amenable to treatment with Formula Icompounds encompass disorders associated with reactions of the specificdefense system as well as with reactions of the nonspecific defensesystem.

“Specific defense system” refers to the component of the immune systemthat reacts to the presence of specific antigens. Examples ofinflammation resulting from a response of the specific defense systeminclude the classical response to foreign antigens, autoimmune diseases,and delayed type hypersensitivity response mediated by T-cells. Chronicinflammatory diseases, the rejection of solid transplanted tissue andorgans, e.g., kidney and bone marrow transplants, and graft versus hostdisease (GVHD), are further examples of inflammatory reactions of thespecific defense system.

The term “nonspecific defense system” as used herein refers toinflammatory disorders that are mediated by leukocytes that areincapable of immunological memory (e.g., granulocytes, and macrophages).Examples of inflammation that result, at least in part, from a reactionof the nonspecific defense system include inflammation associated withconditions such as adult (acute) respiratory distress syndrome (ARDS) ormultiple organ injury syndromes; reperfusion injury; acuteglomerulonephritis; reactive arthritis; dermatoses with acuteinflammatory components; acute purulent meningitis or other centralnervous system inflammatory disorders such as stroke; thermal injury;inflammatory bowel disease; granulocyte transfusion associatedsyndromes; and cytokine-induced toxicity.

“Autoimmune disease” as used herein refers to any group of disorders inwhich tissue injury is associated with humoral or cell-mediatedresponses to the body's own constituents.

“Allergic disease” as used herein refers to any symptoms, tissue damage,or loss of tissue function resulting from allergy. “Arthritic disease”as used herein refers to any disease that is characterized byinflammatory lesions of the joints attributable to a variety ofetiologies. “Dermatitis” as used herein refers to any of a large familyof diseases of the skin that are characterized by inflammation of theskin attributable to a variety of etiologies. “Transplant rejection” asused herein refers to any immune reaction directed against graftedtissue, such as organs or cells (e.g., bone marrow), characterized by aloss of function of the grafted and surrounding tissues, pain, swelling,leukocytosis, and thrombocytopenia. The therapeutic methods of thepresent invention include methods for the treatment of disordersassociated with inflammatory cell activation.

“Inflammatory cell activation” refers to the induction by a stimulus(including, but not limited to, cytokines, antigens or auto-antibodies)of a proliferative cellular response, the production of solublemediators (including but not limited to cytokines, oxygen radicals,enzymes, prostanoids, or vasoactive amines), or cell surface expressionof new or increased numbers of mediators (including, but not limited to,major histocompatability antigens or cell adhesion molecules) ininflammatory cells (including but not limited to monocytes, macrophages,T lymphocytes, B lymphocytes, granulocytes (i.e., polymorphonuclearleukocytes such as neutrophils, basophils, and eosinophils), mast cells,dendritic cells, Langerhans cells, and endothelial cells). It will beappreciated by persons skilled in the art that the activation of one ora combination of these phenotypes in these cells can contribute to theinitiation, perpetuation, or exacerbation of an inflammatory disorder.

The term “NSAID” is an acronym for “non-steroidal anti-inflammatorydrug” and is a therapeutic agent with analgesic, antipyretic (loweringan elevated body temperature and relieving pain without impairingconsciousness) and, in higher doses, with anti-inflammatory effects(reducing inflammation). The term “non-steroidal” is used to distinguishthese drugs from steroids, which (among a broad range of other effects)have a similar eicosanoid-depressing, anti-inflammatory action. Asanalgesics, NSAIDs are unusual in that they are non-narcotic. NSAIDsinclude aspirin, ibuprofen, and naproxen. NSAIDs are usually indicatedfor the treatment of acute or chronic conditions where pain andinflammation are present. NSAIDs are generally indicated for thesymptomatic relief of the following conditions: rheumatoid arthritis,osteoarthritis, inflammatory arthropathies (e.g. ankylosing spondylitis,psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhoea,metastatic bone pain, headache and migraine, postoperative pain,mild-to-moderate pain due to inflammation and tissue injury, pyrexia,ileus, and renal colic. Most NSAIDs act as non-selective inhibitors ofthe enzyme cyclooxygenase, inhibiting both the cyclooxygenase-1 (COX-1)and cyclooxygenase-2 (COX-2) isoenzymes. Cyclooxygenase catalyzes theformation of prostaglandins and thromboxane from arachidonic acid(itself derived from the cellular phospholipid bilayer by phospholipaseA₂). Prostaglandins act (among other things) as messenger molecules inthe process of inflammation. COX-2 inhibitors include celecoxib,etoricoxib, lumiracoxib, parecoxib, rofecoxib, rofecoxib, andvaldecoxib.

The terms “cancer” refers to or describe the physiological condition inmammals that is typically characterized by unregulated cell growth. A“tumor” comprises one or more cancerous cells. Examples of cancerinclude, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia or lymphoid malignancies. More particular examples of suchcancers include squamous cell cancer (e.g., epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-small celllung cancer (“NSCLC”), adenocarcinoma of the lung and squamous carcinomaof the lung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head andneck cancer.

“Hematological malignancies” (British spelling “Haematological”malignancies) are the types of cancer that affect blood, bone marrow,and lymph nodes. As the three are intimately connected through theimmune system, a disease affecting one of the three will often affectthe others as well: although lymphoma is a disease of the lymph nodes,it often spreads to the bone marrow, affecting the blood. Hematologicalmalignancies are malignant neoplasms (“cancer”), and they are generallytreated by specialists in hematology and/or oncology. In some centers“Hematology/oncology” is a single subspecialty of internal medicinewhile in others they are considered separate divisions (there are alsosurgical and radiation oncologists). Not all hematological disorders aremalignant (“cancerous”); these other blood conditions may also bemanaged by a hematologist. Hematological malignancies may derive fromeither of the two major blood cell lineages: myeloid and lymphoid celllines. The myeloid cell line normally produces granulocytes,erythrocytes, thrombocytes, macrophages and mast cells; the lymphoidcell line produces B, T, NK and plasma cells. Lymphomas, lymphocyticleukemias, and myeloma are from the lymphoid line, while acute andchronic myelogenous leukemia, myelodysplastic syndromes andmyeloproliferative diseases are myeloid in origin. Leukemias includeAcute lymphoblastic leukemia (ALL), Acute myelogenous leukemia (AML),Chronic lymphocytic leukemia (CLL), Chronic myelogenous leukemia (CML),Acute monocytic leukemia (AMOL) and small lymphocytic lymphoma (SLL).Lymphomas include Hodgkin's lymphomas (all four subtypes) andNon-Hodgkin's lymphomas (all subtypes).

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer, regardless of mechanism of action. Classes ofchemotherapeutic agents include, but are not limited to: alkylatingagents, antimetabolites, spindle poison plant alkaloids,cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies,photosensitizers, and kinase inhibitors. Chemotherapeutic agents includecompounds used in “targeted therapy” and conventional chemotherapy.Examples of chemotherapeutic agents include: erlotinib (TARCEVA®,Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU(fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®,Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin(cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin(CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology,Princeton, N.J.), trastuzumab (HERCEPTIN®, Genentech), temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide,CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine,NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1/2,HPPD, and rapamycin.

More examples of chemotherapeutic agents include: oxaliplatin(ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent(SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235(PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin(folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib(TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs),gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11,Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, II),vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478,AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib(GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa andcyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, calicheamicin gammaII, calicheamicin omegaI1 (Angew Chem.Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, caminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, nemorubicin,marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin andcarboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide;edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche);ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid; andpharmaceutically acceptable salts, acids and derivatives of any of theabove.

Also included in the definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®;tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifinecitrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrolacetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole,RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX®(anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipidkinase inhibitors; (vi) antisense oligonucleotides, particularly thosewhich inhibit expression of genes in signaling pathways implicated inaberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, suchas oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitorssuch as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptablesalts, acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” aretherapeutic antibodies such as alemtuzumab (Campath), bevacizumab(AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab(VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec),pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®,Genentech), tositumomab (Bexxar, Corixia), and the antibody drugconjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

Humanized monoclonal antibodies with therapeutic potential aschemotherapeutic agents in combination with the Btk inhibitors of theinvention include: alemtuzumab, apolizumab, aselizumab, atlizumab,bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumabmertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab,daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab,fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab,labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab,motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab,ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab,pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab,reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab,sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan,tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab,trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab,urtoxazumab, and visilizumab.

A “metabolite” is a product produced through metabolism in the body of aspecified compound or salt thereof. Metabolites of a compound may beidentified using routine techniques known in the art and theiractivities determined using tests such as those described herein. Suchproducts may result for example from the oxidation, reduction,hydrolysis, amidation, deamidation, esterification, deesterification,enzymatic cleavage, and the like, of the administered compound.Accordingly, the invention includes metabolites of compounds of theinvention, including compounds produced by a process comprisingcontacting a Formula I compound of this invention with a mammal for aperiod of time sufficient to yield a metabolic product thereof.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., NewYork, 1994. The compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention. Many organic compounds exist in opticallyactive forms, i.e., they have the ability to rotate the plane ofplane-polarized light. In describing an optically active compound, theprefixes D and L, or R and S, are used to denote the absoluteconfiguration of the molecule about its chiral center(s). The prefixes dand l or (+) and (−) are employed to designate the sign of rotation ofplane-polarized light by the compound, with (−) or l meaning that thecompound is levorotatory. A compound prefixed with (+) or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity. Enantiomers may be separated from a racemic mixture bya chiral separation method, such as supercritical fluid chromatography(SFC). Assignment of configuration at chiral centers in separatedenantiomers may be tentative, and depicted in Table 1 structures forillustrative purposes, while stereochemical determination awaits, suchas x-ray crystallographic data.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

The term “pharmaceutically acceptable salts” denotes salts which are notbiologically or otherwise undesirable. Pharmaceutically acceptable saltsinclude both acid and base addition salts. The phrase “pharmaceuticallyacceptable” indicates that the substance or composition must becompatible chemically and/or toxicologically, with the other ingredientscomprising a formulation, and/or the mammal being treated therewith.

The term “pharmaceutically acceptable acid addition salt” denotes thosepharmaceutically acceptable salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,carbonic acid, phosphoric acid, and organic acids selected fromaliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,carboxylic, and sulfonic classes of organic acids such as formic acid,acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid,pyruvic acid, oxalic acid, malic acid, maleic acid, malonic acid,succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid,ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamicacid, mandelic acid, embonic acid, phenylacetic acid, methanesulfonicacid “mesylate”, ethanesulfonic acid, p-toluenesulfonic acid, andsalicyclic acid.

The term “pharmaceutically acceptable base addition salt” denotes thosepharmaceutically acceptable salts formed with an organic or inorganicbase. Examples of acceptable inorganic bases include sodium, potassium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, andaluminum salts. Salts derived from pharmaceutically acceptable organicnontoxic bases includes salts of primary, secondary, and tertiaryamines, substituted amines including naturally occurring substitutedamines, cyclic amines and basic ion exchange resins, such asisopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine,dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine,hydrabamine, choline, betaine, ethylenediamine, glucosamine,methylglucamine, theobromine, purines, piperazine, piperidine,N-ethylpiperidine, and polyamine resins

A “solvate” refers to an association or complex of one or more solventmolecules and a compound of the invention. Examples of solvents thatform solvates include, but are not limited to, water, isopropanol,ethanol, methanol, DMSO, ethylacetate, acetic acid, and ethanolamine.

The term “EC₅₀” is the half maximal effective concentration” and denotesthe plasma concentration of a particular compound required for obtaining50% of the maximum of a particular effect in vivo.

The term “Ki” is the inhibition constant and denotes the absolutebinding affinity of a particular inhibitor to a receptor. It is measuredusing competition binding assays and is equal to the concentration wherethe particular inhibitor would occupy 50% of the receptors if nocompeting ligand (e.g. a radioligand) was present. Ki values can beconverted logarithmically to pKi values (−log Ki), in which highervalues indicate exponentially greater potency.

The term “IC₅₀” is the half maximal inhibitory concentration and denotesthe concentration of a particular compound required for obtaining 50%inhibition of a biological process in vitro. IC₅₀ values can beconverted logarithmically to pIC₅₀ values (−log IC₅₀), in which highervalues indicate exponentially greater potency. The IC₅₀ value is not anabsolute value but depends on experimental conditions e.g.concentrations employed, and can be converted to an absolute inhibitionconstant (Ki) using the Cheng-Prusoff equation (Biochem. Pharmacol.(1973) 22:3099). Other percent inhibition parameters, such as IC₇₀,IC₉₀, etc., may be calculated

The terms “compound of this invention,” and “compounds of the presentinvention” and “compounds of Formula I” include compounds of Formulas Iand stereoisomers, geometric isomers, tautomers, solvates, metabolites,and pharmaceutically acceptable salts and prodrugs thereof.

Any formula or structure given herein, including Formula I compounds, isalso intended to represent hydrates, solvates, and polymorphs of suchcompounds, and mixtures thereof.

Any formula or structure given herein, including Formula I compounds, isalso intended to represent unlabeled forms as well as isotopicallylabeled forms of the compounds.

Isotopically labeled compounds have structures depicted by the formulasgiven herein except that one or more atoms are replaced by an atomhaving a selected atomic mass or mass number. Examples of isotopes thatcan be incorporated into compounds of the invention include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine,such as, but not limited to 2H (deuterium, D), 3H (tritium), 11C, 13C,14C, 15N, 18F, 31P, 32P, 35S, 36Cl, and 125I. Various isotopicallylabeled compounds of the present invention, for example those into whichradioactive isotopes such as 3H, 13C, and 14C are incorporated. Suchisotopically labelled compounds may be useful in metabolic studies,reaction kinetic studies, detection or imaging techniques, such aspositron emission tomography (PET) or single-photon emission computedtomography (SPECT) including drug or substrate tissue distributionassays, or in radioactive treatment of patients. Deuterium labelled orsubstituted therapeutic compounds of the invention may have improvedDMPK (drug metabolism and pharmacokinetics) properties, relating todistribution, metabolism, and excretion (ADME). Substitution withheavier isotopes such as deuterium may afford certain therapeuticadvantages resulting from greater metabolic stability, for exampleincreased in vivo half-life or reduced dosage requirements. An 18Flabeled compound may be useful for PET or SPECT studies. Isotopicallylabeled compounds of this invention and prodrugs thereof can generallybe prepared by carrying out the procedures disclosed in the schemes orin the examples and preparations described below by substituting areadily available isotopically labeled reagent for a non-isotopicallylabeled reagent. Further, substitution with heavier isotopes,particularly deuterium (i.e., 2H or D) may afford certain therapeuticadvantages resulting from greater metabolic stability, for exampleincreased in vivo half-life or reduced dosage requirements or animprovement in therapeutic index. It is understood that deuterium inthis context is regarded as a substituent in the compound of the formula(I). The concentration of such a heavier isotope, specificallydeuterium, may be defined by an isotopic enrichment factor. In thecompounds of this invention any atom not specifically designated as aparticular isotope is meant to represent any stable isotope of thatatom. Unless otherwise stated, when a position is designatedspecifically as “H” or “hydrogen”, the position is understood to havehydrogen at its natural abundance isotopic composition. Accordingly, inthe compounds of this invention any atom specifically designated as adeuterium (D) is meant to represent deuterium.

Bicyclic Piperazine Compounds

The present invention provides bicyclic piperazine compounds of FormulaI, including Formulas Ia-Ih, and pharmaceutical formulations thereof,which are potentially useful in the treatment of diseases, conditionsand/or disorders modulated by Btk kinase:

or stereoisomers, tautomers, or pharmaceutically acceptable saltsthereof, wherein:

the solid/dash line

indicates a single or double bond;

X¹ is CR¹ or N;

X² is CR² or N;

X³ is CR³ or N;

where none, one, or two of X¹, X², and X³ are N;

Y¹ and Y² are independently selected from CH and N;

Y³ is C or N;

Y⁴ is CR⁶, N or NH;

where one or two of Y¹, Y², Y³ and Y⁴ are N;

R¹, R² and R³ are independently selected from H, F, Cl, —NH₂, —NHCH₃,—N(CH₃)₂, —OH, —OCH₃, —OCH₂CH₃, —OCH₂CH₂OH, and C₁-C₃ alkyl optionallysubstituted with F, Cl, CN, —NH₂, —NHCH₃, —N(CH₃)₂, —OH, —OCH₃,—OCH₂CH₃, and —OCH₂CH₂OH;

R⁴ is selected from H, F, Cl, CN, —CH₂OH, —CH(CH₃)OH, —C(CH₃)₂OH,—CH(CF₃)OH, —CH₂F, —CHF₂, —CH₂CHF₂, —CF₃, —C(O)NH₂, —C(O)NHCH₃,—C(O)N(CH₃)₂, —NH₂, —NHCH₃, —N(CH₃)₂, —NHC(O)CH₃, —OH, —OCH₃, —OCH₂CH₃,—OCH₂CH₂OH, cyclopropyl, cyclopropylmethyl, 1-hydroxycyclopropyl,imidazolyl, pyrazolyl, 3-hydroxy-oxetan-3-yl, oxetan-3-yl, andazetidin-1-yl;

R⁵ is selected from —CH₃, —CH₂CH₃, —CH₂OH, —CH₂F, —CHF₂, —CF₃, —CN, and—CH₂CH₂OH;

or two R⁵ groups form a 3-, 4-, 5-, or 6-membered carbocyclic orheterocyclic ring;

or an R⁵ group and an R⁸ group form a 3-, 4-, 5-, or 6-memberedcarbocyclic or heterocyclic ring;

n is 0, 1, 2, 3, or 4;

R⁶ is selected from H, Cl, —CH₃, —CH₂CH₃, —CH₂CH₂OH, —CH₂F, —CHF₂, —CF₃,—NH₂, —NHCH₃, —N(CH₃)₂, —OH, —OCH₃, —OCH₂CH₃, and —OCH₂CH₂OH;

R⁷ is selected from the structures:

where the wavy line indicates the site of attachment;

R⁸ is selected from H, —CH₃, —S(O)₂CH₃, cyclopropyl, azetidin-3-yl,oxetan-3-yl, and morpholin-4-yl;

Z is CR⁹ or N; wherein R⁹ is selected from H, F, Cl, —CH₃, —CH₂CH₃,—CH₂CH₂OH, —NH₂, —NHCH₃, —N(CH₃)₂, —OH, —OCH₃, —OCH₂CH₃, and —OCH₂CH₂OH.

Exemplary embodiments of Formula I compounds include compounds ofFormulas Ia-Ih:

Exemplary embodiments of Formula I compounds include wherein X¹ is N, X²is CR², and X³ is CR³.

Exemplary embodiments of Formula I compounds include wherein X¹ is CR¹,X² is N, and X³ is CR³.

Exemplary embodiments of Formula I compounds include wherein X¹ is CR¹,X² is CR², and X³ is N.

Exemplary embodiments of Formula I compounds are selected from: X¹ andX³ are N, X¹ and X² are N, or X² and X³ are N.

Exemplary embodiments of Formula I compounds include wherein R⁴ is—CH₂OH.

Exemplary embodiments of Formula I compounds include wherein X² is CR²,and R² is F.

Exemplary embodiments of Formula I compounds include wherein X¹ and X³are CH.

Exemplary embodiments of Formula I compounds include wherein Y⁴ is CR⁶,and R⁶ is CH₃.

The Formula I compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention.

In addition, the present invention embraces all diastereomers, includingcis-trans (geometric) and conformational isomers. For example, if aFormula I compound incorporates a double bond or a fused ring, the cis-and trans-forms, as well as mixtures thereof, are embraced within thescope of the invention.

In the structures shown herein, where the stereochemistry of anyparticular chiral atom is not specified, then all stereoisomers arecontemplated and included as the compounds of the invention. Wherestereochemistry is specified by a solid wedge or dashed linerepresenting a particular configuration, then that stereoisomer is sospecified and defined.

The compounds of the present invention may exist in unsolvated as wellas solvated forms with pharmaceutically acceptable solvents such aswater, ethanol, and the like, and it is intended that the inventionembrace both solvated and unsolvated forms.

The compounds of the present invention may also exist in differenttautomeric forms, and all such forms are embraced within the scope ofthe invention. The term “tautomer” or “tautomeric form” refers tostructural isomers of different energies which are interconvertible viaa low energy barrier. For example, proton tautomers (also known asprototropic tautomers) include interconversions via migration of aproton, such as keto-enol and imine-enamine isomerizations. Valencetautomers include interconversions by reorganization of some of thebonding electrons.

Biological Evaluation

The relative efficacies of Formula I compounds as inhibitors of anenzyme activity (or other biological activity) can be established bydetermining the concentrations at which each compound inhibits theactivity to a predefined extent and then comparing the results.Typically, the preferred determination is the concentration thatinhibits 50% of the activity in a biochemical assay, i.e., the 50%inhibitory concentration or “IC₅₀”. Determination of IC₅₀ values can beaccomplished using conventional techniques known in the art. In general,an IC₅₀ can be determined by measuring the activity of a given enzyme inthe presence of a range of concentrations of the inhibitor under study.The experimentally obtained values of enzyme activity then are plottedagainst the inhibitor concentrations used. The concentration of theinhibitor that shows 50% enzyme activity (as compared to the activity inthe absence of any inhibitor) is taken as the IC₅₀ value. Analogously,other inhibitory concentrations can be defined through appropriatedeterminations of activity. For example, in some settings it can bedesirable to establish a 90% inhibitory concentration, i.e., IC₉₀, etc.

Formula I compounds were tested by a standard biochemical Btk KinaseAssay (Example 901).

A general procedure for a standard cellular Btk Kinase Assay that can beused to test Formula I compounds is a Ramos Cell Btk Assay (Example902).

A standard cellular B-cell proliferation assay can be used to testFormula I compounds with B-cells purified from spleen of Balb/c mice(Example 903).

A standard T cell proliferation assay can be used to test Formula Icompounds with T-cells purified from spleen of Balb/c mice (Example904).

A CD86 Inhibition assay can be conducted on Formula I compounds for theinhibition of B cell activity using total mouse splenocytes purifiedfrom spleens of 8-16 week old Balb/c mice (Example 905).

A B-ALL Cell Survival Assay can be conducted on Formula I compounds tomeasure the number of viable B-ALL cells in culture (Example 906).

A CD69 Whole Blood Assay can be conducted on Formula I compounds todetermine the ability of compounds to inhibit the production of CD69 byB lymphocytes in human whole blood activated by crosslinking surface IgMwith goat F(ab′)2 anti-human IgM (Example 907). CD69 is a type II C-typelectin involved in lymphocyte migration and cytokine secretion. CD69expression represents one of the earliest available indicators ofleukocyte activation and its rapid induction occurs throughtranscriptional activation (Vazquez et al (2009) Jour. of ImmunologyPublished Oct. 19, 2009, doi:10.4049/jimmunol.0900839).Concentration-dependent inhibition of antigen receptor stimulation byselective Btk inhibitors induces cell surface expression of thelymphocyte activation marker CD69 (Honigberg et al (2010) Proc. Natl.Acad. Sci. 107(29):13075-13080). Thus, CD69 inhibition by selective Btkinhibitors may be correlated with therapeutic efficacy of certain B-celldisorders. The CD69 Hu Blood FACS IC70 values are displayed forexemplary Formula I compounds in Tables 1 and 2.

The cytotoxic or cytostatic activity of Formula I exemplary compoundscan be measured by: establishing a proliferating mammalian tumor cellline in a cell culture medium, adding a Formula I compound, culturingthe cells for a period from about 6 hours to about 5 days; and measuringcell viability (Example 908). Cell-based in vitro assays are used tomeasure viability, i.e. proliferation (IC₅₀), cytotoxicity (EC₅₀), andinduction of apoptosis (caspase activation) and may be useful inpredicting clinical efficacy against hematological malignancies andsolid tumors.

The in vitro potency of the combinations of Formula I compounds withchemotherapeutic agents can be measured by the cell proliferation assayof Example 908; the CellTiter-Glo® Luminescent Cell Viability Assay,commercially available from Promega Corp., Madison, Wis. Thishomogeneous assay method is based on the recombinant expression ofColeoptera luciferase (U.S. Pat. No. 5,583,024; U.S. Pat. No. 5,674,713;U.S. Pat. No. 5,700,670) and determines the number of viable cells inculture based on quantitation of the ATP present, an indicator ofmetabolically active cells (Crouch et al (1993) J. Immunol. Meth.160:81-88; U.S. Pat. No. 6,602,677). The CellTiter-Glo® Assay wasconducted in 96 or 384 well format, making it amenable to automatedhigh-throughput screening (HTS) (Cree et al (1995) AntiCancer Drugs6:398-404). The homogeneous assay procedure involves adding the singlereagent (CellTiter-Glo® Reagent) directly to cells cultured inserum-supplemented medium. Cell washing, removal of medium and multiplepipetting steps are not required. The system detects as few as 15cells/well in a 384-well format in 10 minutes after adding reagent andmixing.

The homogeneous “add-mix-measure” format results in cell lysis andgeneration of a luminescent signal proportional to the amount of ATPpresent. The amount of ATP is directly proportional to the number ofcells present in culture. The CellTiter-Glo® Assay generates a“glow-type” luminescent signal, produced by the luciferase reaction,which has a half-life generally greater than five hours, depending oncell type and medium used. Viable cells are reflected in relativeluminescence units (RLU). The substrate, Beetle Luciferin, isoxidatively decarboxylated by recombinant firefly luciferase withconcomitant conversion of ATP to AMP and generation of photons. Theextended half-life eliminates the need to use reagent injectors andprovides flexibility for continuous or batch mode processing of multipleplates. This cell proliferation assay can be used with various multiwellformats, e.g. 96 or 384 well format. Data can be recorded by luminometeror CCD camera imaging device. The luminescence output is presented asrelative light units (RLU), measured over time.

The anti-proliferative efficacy of Formula I exemplary compounds andcombinations with chemotherapeutic agents are measured by theCellTiter-Glo® Assay (Example 908) against certain hematological tumorcell lines. EC₅₀ values are established for the tested compounds andcombinations.

Exemplary Formula I compounds in Tables 1 and 2 were made,characterized, and tested for inhibition of Btk according to the methodsof this invention, and have the following structures and correspondingnames (ChemDraw Ultra, Version 9.0.1, and ChemBioDraw, Version 11.0,CambridgeSoft Corp., Cambridge Mass.). Where more than one name isassociated with a Formula I compound or intermediate, the chemicalstructure shall define the compound.

TABLE 1 CD69 Hu Blood Mol FACS No. Structure IUPAC_Name Weight IC70 101

2-(5-fluoro-2- (hydroxymethyl)-3-(8-(5-(4- methylpiperazin-1-yl)pyridin-2- ylamino)imidazo[1,2- a]pyridin-6-yl)phenyl)- 3,4,6,7,8,9-hexahydropyrazino[1,2- a]indol-1(2H)-one 621 0.007 102

2-(5-fluoro-2- (hydroxymethyl)-3-(8-(5-(4- (oxetan-3-yl)piperazin-1-yl)pyridine-2- ylamino)imidazo[1,2- a]pyridin-6-yl)phenyl)- 3,4,6,7,8,9-hexahydropyrazino[1,2- a]indol-1(2H)-one 663 0.011 103

5-[5-fluoro-2- (hydroxymethyl)-3-{(8-(5-(4- methylpiperazin-1-yl)pyridin-2- ylamino)imidazo[1,2- a]pyridin-6-yl)}phenyl]-8-thia-5-azatricyclo- [7.4.0.0^(2,7)]trideca-1(9),2(7)- dien-6-one 6380.319 104

2-(5-fluoro-2- (hydroxymethyl)-3-(8-(5-(4- methylpiperazin-1-yl)pyridin-2- ylamino)imidazo[1,2- b]pyridazin-6-yl)phenyl)-3,4,6,7,8,9- hexahydropyrazino[1,2- a]indol-1(2H)-one 622 0.096 105

2-(5-fluoro-2- (hydroxymethyl)-3-(8-(5-(4- (oxetan-3-yl)piperazin-1-yl)pyridin-2- ylamino)imidazo[1,2- b]pyridazin-6-yl)phenyl)-3,4,6,7,8,9- hexahydropyrazino[1,2- a]indol-1(2H)-one 664 0.049 106

2-(5-fluoro-2- (hydroxymethyl)-3-(1- methyl-5-(5-(4-(oxetan-3-yl)-1,4-diazepan-1-yl)pyridin-2- ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)- 3,4,6,7,8,9- hexahydropyrazino[1,2-a]indol-1(2H)-one 622 0.814 107

2-(5-fluoro-2- (hydroxymethyl)-3-(8-(5-(4- (oxetan-3-yl)piperazin-1-yl)pyridin- 2ylamino)imidazo[1,2- a]pyrazin-6-yl)phenyl)- 3,4,6,7,8,9-hexahydropyrazino[1,2- a]indol-1(2H)-one 664.3 0.939 108

2-(5-fluoro-2- (hydroxymethyl)-3-(8-(5-(4- (oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)- [1,2,4]triazolo[1,5-a]pyridin-6-yl)phenyl)-3,4,6,7,8,9- hexahydropyrazino[1,2- a]indol-1(2H)-one 6640.058 109

2-(5-fluoro-2- (hydroxymethyl)-3-(3- methyl-7-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2- ylamino)-3H- benzo[d]imidazol-5-yl)phenyl)-3,4,6,7,8,9- hexahydropyrazino[1,2- a]indol-1(2H)-one 677.31.3 110

10-[5-fluoro-2- (hydroxymethyl)-3-(8-(5-(4- (oxetan-3-yl)piperazin-1-yl)pyridin-2- ylamino)imidazo[1,2- b]pyridazin-6-yl)phenyl]-4,4-dimethyl-1,10- diazatricyclo[6.4.0.0^(2,6)]- dodeca-2(6),7-dien-9-one678 0.015 111

5-[5-fluoro-2- (hydroxymethyl)-3-(8-(5-(4- (oxetan-3-yl)piperazin-1-yl)pyridin-2- ylamino)imidazo[1,2- b]pyridazin-6-yl)phenyl]-8-thia-5-azatricyclo- [7.4.0.0^(2,7)]trideca-1(9),2(7)- dien-6-one 6810.313 112

5-[5-fluoro-2- (hydroxymethyl)-3-(8-(5-(4- (oxetan-3-yl)piperazin-1-yl)pyridin-2- ylamino)imidazo[1,2- b]pyridazin-6-yl)phenyl]-8-thia-4,5-diazatricyclo- [7.4.0.0^(2,7)]trideca-1(9),2(7),3- trien-6-one680 0.126 113

10-[5-fluoro-2- (hydroxymethyl)-3-(8-(5-(4- (oxetan-3-yl)piperazin-1-yl)pyridin-2- ylamino)imidazo[1,2- b]pyridazin-6-yl)phenyl]-4,4-dimethyl-7-thia-10- azatricyclo[6.4.0.0^(2,6)]dodeca-1(8),2(6)-dien-9-one 695 0.012 114

2-(3-(hydroxymethyl)-4-(8- (5-(4-(oxetan-3-yl)piperazin- 1-yl)pyridin-2-ylamino)imidazo[1,2- b]pyridazin-6-yl)pyridin-2- yl)-3,4,6,7,8,9-hexahydropyrazino[1,2- a]indol-1(2H)-one 647 0.118 115

(S)-2-(3-(hydroxymethyl)-4- (8-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2- ylamino)imidazo[1,2-b]pyridazin-6-yl)pyridin-2- yl)-3,4,6,7,8,9- hexahydropyrazino[1,2-a]indol-1(2H)-one 660.33

TABLE 2 CD69 Hu Blood FACS No. Structure IUPAC_Name (IC70) 116

2-[5-fluoro-2-(hydroxymethyl)- 3-[7-[[5-[4-(oxetan-3-yl)piperazin-1-yl]-2- pyridyl]amino]-3H- benzimidazol-5-yl]phenyl]-3,4,6,7,8,9- hexahydropyrazino[1,2-a]indol- 1-one 1.3 117

2-[5-fluoro-2-(hydroxymethyl)- 3-[6-[4-[4-(oxetan-3-yl)piperazin-1-yl]anilino]-9H- purin-2-yl]phenyl]-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol- 1-one 0.812 118

2-[3-(hydroxymethyl)-4-[7-[[5- [4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-3H- benzimidazol-5-yl]-2-pyridyl]- 3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol- 1-one 4.3 119

2-[5-fluoro-2-(hydroxymethyl)- 3-[6-[4-[4-(oxetan-3-yl)piperazin-1-yl]anilino]-9H- purin-2-yl]phenyl]-3,4,5,6,7,8-hexahydrobenzothiopheno[2,3- c]pyridin-1-one 0.727 120

2-[5-fluoro-2-(hydroxymethyl)- 3-[6-[4-[4-(oxetan-3-yl)piperazin-1-yl]anilino]-9H- purin-2-yl]phenyl]-3,4,6,7,8,9-hexahydropyrido[3,4- b]indolizin-1-one 1.9 121

2-[3-(hydroxymethyl)-4-[8-[[5- [4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]imidazo[1,2- a]pyridin-6-yl]-2-pyridyl]- 3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol- 1-one 0.279 122

2-[3-(hydroxymethyl)-4-[8-[[5- [4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]- [1,2,4]triazolo[1,5-a]pyridin-6-yl]-2-pyridyl]-3,4,6,7,8,9- hexahydropyrazino[1,2-a]indol- 1-one 0.208123

3-[5-fluoro-2-(hydroxymethyl)- 3-[6-[4-[4-(oxetan-3-yl)piperazin-1-yl]anilino]-9H- purin-2-yl]phenyl]-7,7- dimethyl-1,2,6,8-tetrahydrocyclopenta[3,4]- pyrrolo[3,5-b]pyrazin-4-one 0.256 124

2-[3-(hydroxymethyl)-4-[8-[(5- methyl-6,7-dihydro-4H-pyrazolo[1,5-a]pyrazin-2- yl)amino]imidazo[1,2-b]pyridazin-6-yl]-2-pyridyl]- 3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol- 1-one 0.333Administration of Formula I Compounds

The compounds of the invention may be administered by any routeappropriate to the condition to be treated. Suitable routes includeoral, parenteral (including subcutaneous, intramuscular, intravenous,intraarterial, intradermal, intrathecal and epidural), transdermal,rectal, nasal, topical (including buccal and sublingual), vaginal,intraperitoneal, intrapulmonary and intranasal. For localimmunosuppressive treatment, the compounds may be administered byintralesional administration, including perfusing or otherwisecontacting the graft with the inhibitor before transplantation. It willbe appreciated that the preferred route may vary with for example thecondition of the recipient. Where the compound is administered orally,it may be formulated as a pill, capsule, tablet, etc. with apharmaceutically acceptable carrier or excipient. Where the compound isadministered parenterally, it may be formulated with a pharmaceuticallyacceptable parenteral vehicle and in a unit dosage injectable form, asdetailed below.

A dose to treat human patients may range from about 10 mg to about 1000mg of Formula I compound. A typical dose may be about 100 mg to about300 mg of the compound. A dose may be administered once a day (QID),twice per day (BID), or more frequently, depending on thepharmacokinetic and pharmacodynamic properties, including absorption,distribution, metabolism, and excretion of the particular compound. Inaddition, toxicity factors may influence the dosage and administrationregimen. When administered orally, the pill, capsule, or tablet may beingested daily or less frequently for a specified period of time. Theregimen may be repeated for a number of cycles of therapy.

Methods of Treatment with Formula I Compounds

Formula I compounds of the present invention are useful for treating ahuman or animal patient suffering from a disease or disorder arisingfrom abnormal cell growth, function or behavior associated with Btkkinase such as an immune disorder, cardiovascular disease, viralinfection, inflammation, a metabolism/endocrine disorder or aneurological disorder, may thus be treated by a method comprising theadministration thereto of a compound of the present invention as definedabove. A human or animal patient suffering from cancer may also betreated by a method comprising the administration thereto of a compoundof the present invention as defined above. The condition of the patientmay thereby be improved or ameliorated.

Formula I compounds may be useful for in vitro, in situ, and in vivodiagnosis or treatment of mammalian cells, organisms, or associatedpathological conditions, such as systemic and local inflammation,immune-inflammatory diseases such as rheumatoid arthritis, immunesuppression, organ transplant rejection, allergies, ulcerative colitis,Crohn's disease, dermatitis, asthma, systemic lupus erythematosus,Sjögren's Syndrome, multiple sclerosis, scleroderma/systemic sclerosis,idiopathic thrombocytopenic purpura (ITP), anti-neutrophil cytoplasmicantibodies (ANCA) vasculitis, chronic obstructive pulmonary disease(COPD), psoriasis, and for general joint protective effects.

Methods of the invention also include treating such diseases asarthritic diseases, such as rheumatoid arthritis, monoarticulararthritis, osteoarthritis, gouty arthritis, spondylitis; Behcet disease;sepsis, septic shock, endotoxic shock, gram negative sepsis, grampositive sepsis, and toxic shock syndrome; multiple organ injurysyndrome secondary to septicemia, trauma, or hemorrhage; ophthalmicdisorders such as allergic conjunctivitis, vernal conjunctivitis,uveitis, and thyroid-associated ophthalmopathy; eosinophilic granuloma;pulmonary or respiratory disorders such as asthma, chronic bronchitis,allergic rhinitis, ARDS, chronic pulmonary inflammatory disease (e.g.,chronic obstructive pulmonary disease), silicosis, pulmonarysarcoidosis, pleurisy, alveolitis, vasculitis, emphysema, pneumonia,bronchiectasis, and pulmonary oxygen toxicity; reperfusion injury of themyocardium, brain, or extremities; fibrosis such as cystic fibrosis;keloid formation or scar tissue formation; atherosclerosis; autoimmunediseases, such as systemic lupus erythematosus (SLE), autoimmunethyroiditis, multiple sclerosis, some forms of diabetes, and Reynaud'ssyndrome; and transplant rejection disorders such as GVHD and allograftrejection; chronic glomerulonephritis; inflammatory bowel diseases suchas chronic inflammatory bowel disease (CIBD), Crohn's disease,ulcerative colitis, and necrotizing enterocolitis; inflammatorydermatoses such as contact dermatitis, atopic dermatitis, psoriasis, orurticaria; fever and myalgias due to infection; central or peripheralnervous system inflammatory disorders such as meningitis, encephalitis,and brain or spinal cord injury due to minor trauma; Sjogren's syndrome;diseases involving leukocyte diapedesis; alcoholic hepatitis; bacterialpneumonia; antigen-antibody complex mediated diseases; hypovolemicshock; Type I diabetes mellitus; acute and delayed hypersensitivity;disease states due to leukocyte dyscrasia and metastasis; thermalinjury; granulocyte transfusion-associated syndromes; andcytokine-induced toxicity.

Methods of the invention also include treating cancer selected frombreast, ovary, cervix, prostate, testis, genitourinary tract, esophagus,larynx, glioblastoma, neuroblastoma, stomach, skin, keratoacanthoma,lung, epidermoid carcinoma, large cell carcinoma, non-small cell lungcarcinoma (NSCLC), small cell carcinoma, lung adenocarcinoma, bone,colon, adenoma, pancreas, adenocarcinoma, thyroid, follicular carcinoma,undifferentiated carcinoma, papillary carcinoma, seminoma, melanoma,sarcoma, bladder carcinoma, liver carcinoma and biliary passages, kidneycarcinoma, pancreatic, myeloid disorders, lymphoma, hairy cells, buccalcavity, naso-pharyngeal, pharynx, lip, tongue, mouth, small intestine,colon-rectum, large intestine, rectum, brain and central nervous system,Hodgkin's, leukemia, bronchus, thyroid, liver and intrahepatic bileduct, hepatocellular, gastric, glioma/glioblastoma, endometrial,melanoma, kidney and renal pelvis, urinary bladder, uterine corpus,uterine cervix, multiple myeloma, acute myelogenous leukemia, chronicmyelogenous leukemia, lymphocytic leukemia, chronic lymphoid leukemia(CLL), myeloid leukemia, oral cavity and pharynx, non-Hodgkin lymphoma,melanoma, and villous colon adenoma.

The methods of the invention can have utility in treating subjects whoare or can be subject to reperfusion injury, i.e., injury resulting fromsituations in which a tissue or organ experiences a period of ischemiafollowed by reperfusion. The term “ischemia” refers to localized tissueanemia due to obstruction of the inflow of arterial blood. Transientischemia followed by reperfusion characteristically results inneutrophil activation and transmigration through the endothelium of theblood vessels in the affected area. Accumulation of activatedneutrophils in turn results in generation of reactive oxygenmetabolites, which damage components of the involved tissue or organ.This phenomenon of “reperfusion injury” is commonly associated withconditions such as vascular stroke (including global and focalischemia), hemorrhagic shock, myocardial ischemia or infarction, organtransplantation, and cerebral vasospasm. To illustrate, reperfusioninjury occurs at the termination of cardiac bypass procedures or duringcardiac arrest when the heart, once prevented from receiving blood,begins to reperfuse. It is expected that inhibition of Btk activity mayresult in reduced amounts of reperfusion injury in such situations.

Pharmaceutical Formulations

In order to use a compound of this invention for the therapeutictreatment of mammals including humans, it is normally formulated inaccordance with standard pharmaceutical practice as a pharmaceuticalcomposition. According to this aspect of the invention there is provideda pharmaceutical composition comprising a compound of this invention inassociation with a pharmaceutically acceptable diluent or carrier.

A typical formulation is prepared by mixing a compound of the presentinvention and a carrier, diluent or excipient. Suitable carriers,diluents and excipients are well known to those skilled in the art andinclude materials such as carbohydrates, waxes, water soluble and/orswellable polymers, hydrophilic or hydrophobic materials, gelatin, oils,solvents, water and the like. The particular carrier, diluent orexcipient used will depend upon the means and purpose for which thecompound of the present invention is being applied. Solvents aregenerally selected based on solvents recognized by persons skilled inthe art as safe (GRAS) to be administered to a mammal. In general, safesolvents are non-toxic aqueous solvents such as water and othernon-toxic solvents that are soluble or miscible in water. Suitableaqueous solvents include water, ethanol, propylene glycol, polyethyleneglycols (e.g., PEG 400, PEG 300), etc. and mixtures thereof. Theformulations may also include one or more buffers, stabilizing agents,surfactants, wetting agents, lubricating agents, emulsifiers, suspendingagents, preservatives, antioxidants, opaquing agents, glidants,processing aids, colorants, sweeteners, perfuming agents, flavoringagents and other known additives to provide an elegant presentation ofthe drug (i.e., a compound of the present invention or pharmaceuticalcomposition thereof) or aid in the manufacturing of the pharmaceuticalproduct (i.e., medicament).

The formulations may be prepared using conventional dissolution andmixing procedures. For example, the bulk drug substance (i.e., compoundof the present invention or stabilized form of the compound (e.g.,complex with a cyclodextrin derivative or other known complexationagent) is dissolved in a suitable solvent in the presence of one or moreof the excipients described above. The compound of the present inventionis typically formulated into pharmaceutical dosage forms to provide aneasily controllable dosage of the drug and to enable patient compliancewith the prescribed regimen.

The pharmaceutical composition (or formulation) for application may bepackaged in a variety of ways depending upon the method used foradministering the drug. Generally, an article for distribution includesa container having deposited therein the pharmaceutical formulation inan appropriate form. Suitable containers are well known to those skilledin the art and include materials such as bottles (plastic and glass),sachets, ampoules, plastic bags, metal cylinders, and the like. Thecontainer may also include a tamper-proof assemblage to preventindiscreet access to the contents of the package. In addition, thecontainer has deposited thereon a label that describes the contents ofthe container. The label may also include appropriate warnings.

Pharmaceutical formulations of the compounds of the present inventionmay be prepared for various routes and types of administration. Forexample, a compound of Formula I having the desired degree of purity mayoptionally be mixed with pharmaceutically acceptable diluents, carriers,excipients or stabilizers (Remington's Pharmaceutical Sciences (1980)16th edition, Osol, A. Ed.), in the form of a lyophilized formulation,milled powder, or an aqueous solution. Formulation may be conducted bymixing at ambient temperature at the appropriate pH, and at the desireddegree of purity, with physiologically acceptable carriers, i.e.,carriers that are non-toxic to recipients at the dosages andconcentrations employed. The pH of the formulation depends mainly on theparticular use and the concentration of compound, but may range fromabout 3 to about 8. Formulation in an acetate buffer at pH 5 is asuitable embodiment.

The compound ordinarily can be stored as a solid composition, alyophilized formulation or as an aqueous solution.

The pharmaceutical compositions of the invention will be formulated,dosed and administered in a fashion, i.e., amounts, concentrations,schedules, course, vehicles and route of administration, consistent withgood medical practice. Factors for consideration in this context includethe particular disorder being treated, the particular mammal beingtreated, the clinical condition of the individual patient, the cause ofthe disorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The “therapeutically effective amount”of the compound to be administered will be governed by suchconsiderations, and is the minimum amount necessary to ameliorate, ortreat the hyperproliferative disorder.

As a general proposition, the initial pharmaceutically effective amountof the inhibitor administered parenterally per dose will be in the rangeof about 0.01-100 mg/kg, namely about 0.1 to 20 mg/kg of patient bodyweight per day, with the typical initial range of compound used being0.3 to 15 mg/kg/day.

Acceptable diluents, carriers, excipients and stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Theactive pharmaceutical ingredients may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations of compounds of Formula I may beprepared. Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing acompound of Formula I, which matrices are in the form of shapedarticles, e.g., films, or microcapsules. Examples of sustained-releasematrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate) and poly-D-(−)-3-hydroxybutyric acid.

The formulations include those suitable for the administration routesdetailed herein. The formulations may conveniently be presented in unitdosage form and may be prepared by any of the methods well known in theart of pharmacy. Techniques and formulations generally are found inRemington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.).Such methods include the step of bringing into association the activeingredient with the carrier which constitutes one or more accessoryingredients. In general the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

Formulations of a compound of Formula I suitable for oral administrationmay be prepared as discrete units such as pills, capsules, cachets ortablets each containing a predetermined amount of a compound of FormulaI. Compressed tablets may be prepared by compressing in a suitablemachine the active ingredient in a free-flowing form such as a powder orgranules, optionally mixed with a binder, lubricant, inert diluent,preservative, surface active or dispersing agent. Molded tablets may bemade by molding in a suitable machine a mixture of the powdered activeingredient moistened with an inert liquid diluent. The tablets mayoptionally be coated or scored and optionally are formulated so as toprovide slow or controlled release of the active ingredient therefrom.Tablets, troches, lozenges, aqueous or oil suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, e.g., gelatincapsules, syrups or elixirs may be prepared for oral use. Formulationsof compounds of Formula I intended for oral use may be preparedaccording to any method known to the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents including sweetening agents, flavoring agents, coloringagents and preserving agents, in order to provide a palatablepreparation. Tablets containing the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipient which are suitable formanufacture of tablets are acceptable. These excipients may be, forexample, inert diluents, such as calcium or sodium carbonate, lactose,calcium or sodium phosphate; granulating and disintegrating agents, suchas maize starch, or alginic acid; binding agents, such as starch,gelatin or acacia; and lubricating agents, such as magnesium stearate,stearic acid or talc. Tablets may be uncoated or may be coated by knowntechniques including microencapsulation to delay disintegration andadsorption in the gastrointestinal tract and thereby provide a sustainedaction over a longer period. For example, a time delay material such asglyceryl monostearate or glyceryl distearate alone or with a wax may beemployed.

For treatment of the eye or other external tissues, e.g., mouth andskin, the formulations are preferably applied as a topical ointment orcream containing the active ingredient(s) in an amount of, for example,0.075 to 20% w/w. When formulated in an ointment, the active ingredientsmay be employed with either a paraffinic or a water-miscible ointmentbase. Alternatively, the active ingredients may be formulated in a creamwith an oil-in-water cream base. If desired, the aqueous phase of thecream base may include a polyhydric alcohol, i.e., an alcohol having twoor more hydroxyl groups such as propylene glycol, butane 1,3-diol,mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400)and mixtures thereof. The topical formulations may desirably include acompound which enhances absorption or penetration of the activeingredient through the skin or other affected areas. Examples of suchdermal penetration enhancers include dimethyl sulfoxide and relatedanalogs. The oily phase of the emulsions of this invention may beconstituted from known ingredients in a known manner. While the phasemay comprise merely an emulsifier, it desirably comprises a mixture ofat least one emulsifier with a fat or an oil or with both a fat and anoil. Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier which acts as a stabilizer. It is also preferredto include both an oil and a fat. Together, the emulsifier(s) with orwithout stabilizer(s) make up the so-called emulsifying wax, and the waxtogether with the oil and fat make up the so-called emulsifying ointmentbase which forms the oily dispersed phase of the cream formulations.Emulsifiers and emulsion stabilizers suitable for use in the formulationof the invention include Tween® 60, Span® 80, cetostearyl alcohol,benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodiumlauryl sulfate.

Aqueous suspensions of Formula I compounds contain the active materialsin admixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, croscarmellose, povidone, methylcellulose,hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone,gum tragacanth and gum acacia, and dispersing or wetting agents such asa naturally occurring phosphatide (e.g., lecithin), a condensationproduct of an alkylene oxide with a fatty acid (e.g., polyoxyethylenestearate), a condensation product of ethylene oxide with a long chainaliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensationproduct of ethylene oxide with a partial ester derived from a fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). Theaqueous suspension may also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such as sucroseor saccharin.

The pharmaceutical compositions of compounds of Formula I may be in theform of a sterile injectable preparation, such as a sterile injectableaqueous or oleaginous suspension. This suspension may be formulatedaccording to the known art using those suitable dispersing or wettingagents and suspending agents which have been mentioned above. Thesterile injectable preparation may also be a sterile injectable solutionor suspension in a non-toxic parenterally acceptable diluent or solvent,such as a solution in 1,3-butanediol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, atime-release formulation intended for oral administration to humans maycontain approximately 1 to 1000 mg of active material compounded with anappropriate and convenient amount of carrier material which may varyfrom about 5 to about 95% of the total compositions (weight:weight). Thepharmaceutical composition can be prepared to provide easily measurableamounts for administration. For example, an aqueous solution intendedfor intravenous infusion may contain from about 3 to 500 μg of theactive ingredient per milliliter of solution in order that infusion of asuitable volume at a rate of about 30 mL/hr can occur.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the activeingredient. The active ingredient is preferably present in suchformulations in a concentration of about 0.5 to 20% w/w, for exampleabout 0.5 to 10% w/w, for example about 1.5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for intrapulmonary or nasal administration have aparticle size for example in the range of 0.1 to 500 microns (includingparticle sizes in a range between 0.1 and 500 microns in incrementsmicrons such as 0.5, 1, 30 microns, 35 microns, etc.), which isadministered by rapid inhalation through the nasal passage or byinhalation through the mouth so as to reach the alveolar sacs. Suitableformulations include aqueous or oily solutions of the active ingredient.Formulations suitable for aerosol or dry powder administration may beprepared according to conventional methods and may be delivered withother therapeutic agents such as compounds heretofore used in thetreatment or prophylaxis disorders as described below.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

The formulations may be packaged in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water, for injection immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Preferred unit dosage formulations are thosecontaining a daily dose or unit daily sub-dose, as herein above recited,or an appropriate fraction thereof, of the active ingredient.

The invention further provides veterinary compositions comprising atleast one active ingredient as above defined together with a veterinarycarrier therefore. Veterinary carriers are materials useful for thepurpose of administering the composition and may be solid, liquid orgaseous materials which are otherwise inert or acceptable in theveterinary art and are compatible with the active ingredient. Theseveterinary compositions may be administered parenterally, orally or byany other desired route.

Combination Therapy

The compounds of Formula I may be employed alone or in combination withother therapeutic agents for the treatment of a disease or disorderdescribed herein, such as inflammation or a hyperproliferative disorder(e.g., cancer). In certain embodiments, a compound of Formula I iscombined in a pharmaceutical combination formulation, or dosing regimenas combination therapy, with an additional, second therapeutic compoundthat has anti-inflammatory or anti-hyperproliferative properties or thatis useful for treating an inflammation, immune-response disorder, orhyperproliferative disorder (e.g., cancer). The additional therapeuticmay be an anti-inflammatory agent, an immunomodulatory agent,chemotherapeutic agent, an apoptosis-enhancer, a neurotropic factor, anagent for treating cardiovascular disease, an agent for treating liverdisease, an anti-viral agent, an agent for treating blood disorders, anagent for treating diabetes, and an agent for treating immunodeficiencydisorders. The second therapeutic agent may be an NSAIDanti-inflammatory agent. The second therapeutic agent may be achemotherapeutic agent. The second compound of the pharmaceuticalcombination formulation or dosing regimen preferably has complementaryactivities to the compound of Formula I such that they do not adverselyaffect each other. Such compounds are suitably present in combination inamounts that are effective for the purpose intended. In one embodiment,a composition of this invention comprises a compound of Formula I, or astereoisomer, tautomer, solvate, metabolite, or pharmaceuticallyacceptable salt or prodrug thereof, in combination with a therapeuticagent such as an NSAID.

The combination therapy may be administered as a simultaneous orsequential regimen. When administered sequentially, the combination maybe administered in two or more administrations. The combinedadministration includes coadministration, using separate formulations ora single pharmaceutical formulation, and consecutive administration ineither order, wherein preferably there is a time period while both (orall) active agents simultaneously exert their biological activities.

Suitable dosages for any of the above coadministered agents are thosepresently used and may be lowered due to the combined action (synergy)of the newly identified agent and other therapeutic agents ortreatments.

The combination therapy may provide “synergy” and prove “synergistic”,i.e., the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect may be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect may be attained when the compounds are administered or deliveredsequentially, e.g., by different injections in separate syringes,separate pills or capsules, or separate infusions. In general, duringalternation therapy, an effective dosage of each active ingredient isadministered sequentially, i.e., serially, whereas in combinationtherapy, effective dosages of two or more active ingredients areadministered together.

In a particular embodiment of therapy, a compound of Formula I, or astereoisomer, tautomer, solvate, metabolite, or pharmaceuticallyacceptable salt or prodrug thereof, may be combined with othertherapeutic, hormonal or antibody agents such as those described herein,as well as combined with surgical therapy and radiotherapy. Combinationtherapies according to the present invention thus comprise theadministration of at least one compound of Formula I, or a stereoisomer,tautomer, solvate, metabolite, or pharmaceutically acceptable salt orprodrug thereof, and the use of at least one other cancer treatmentmethod. The amounts of the compound(s) of Formula I and the otherpharmaceutically active therapeutic agent(s) and the relative timings ofadministration will be selected in order to achieve the desired combinedtherapeutic effect.

Metabolites of Compounds of Formula I

Also falling within the scope of this invention are the in vivometabolic products of Formula I described herein. Such products mayresult for example from the oxidation, reduction, hydrolysis, amidation,deamidation, esterification, deesterification, enzymatic cleavage, andthe like, of the administered compound. Accordingly, the inventionincludes metabolites of compounds of Formula I, including compoundsproduced by a process comprising contacting a compound of this inventionwith a mammal for a period of time sufficient to yield a metabolicproduct thereof.

Metabolite products typically are identified by preparing aradiolabelled (e.g., ¹⁴C or ³H) isotope of a compound of the invention,administering it parenterally in a detectable dose (e.g., greater thanabout 0.5 mg/kg) to an animal such as rat, mouse, guinea pig, monkey, orto man, allowing sufficient time for metabolism to occur (typicallyabout 30 seconds to 30 hours) and isolating its conversion products fromthe urine, blood or other biological samples. These products are easilyisolated since they are labeled (others are isolated by the use ofantibodies capable of binding epitopes surviving in the metabolite). Themetabolite structures are determined in conventional fashion, e.g., byMS, LC/MS or NMR analysis. In general, analysis of metabolites is donein the same way as conventional drug metabolism studies well known tothose skilled in the art. The metabolite products, so long as they arenot otherwise found in vivo, are useful in diagnostic assays fortherapeutic dosing of the compounds of the invention.

Articles of Manufacture

In another embodiment of the invention, an article of manufacture, or“kit”, containing materials useful for the treatment of the diseases anddisorders described above is provided. In one embodiment, the kitcomprises a container comprising a compound of Formula I, or astereoisomer, tautomer, solvate, metabolite, or pharmaceuticallyacceptable salt or prodrug thereof. The kit may further comprise a labelor package insert on or associated with the container. The term “packageinsert” is used to refer to instructions customarily included incommercial packages of therapeutic products, that contain informationabout the indications, usage, dosage, administration, contraindicationsand/or warnings concerning the use of such therapeutic products.Suitable containers include, for example, bottles, vials, syringes,blister pack, etc. The container may be formed from a variety ofmaterials such as glass or plastic. The container may hold a compound ofFormula I or a formulation thereof which is effective for treating thecondition and may have a sterile access port (for example, the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is a compound of Formula I. The label or package insertindicates that the composition is used for treating the condition ofchoice, such as cancer. In addition, the label or package insert mayindicate that the patient to be treated is one having a disorder such asa hyperproliferative disorder, neurodegeneration, cardiac hypertrophy,pain, migraine or a neurotraumatic disease or event. In one embodiment,the label or package inserts indicates that the composition comprising acompound of Formula I can be used to treat a disorder resulting fromabnormal cell growth. The label or package insert may also indicate thatthe composition can be used to treat other disorders. Alternatively, oradditionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

The kit may further comprise directions for the administration of thecompound of Formula I and, if present, the second pharmaceuticalformulation. For example, if the kit comprises a first compositioncomprising a compound of Formula I and a second pharmaceuticalformulation, the kit may further comprise directions for thesimultaneous, sequential or separate administration of the first andsecond pharmaceutical compositions to a patient in need thereof.

In another embodiment, the kits are suitable for the delivery of solidoral forms of a compound of Formula I, such as tablets or capsules. Sucha kit preferably includes a number of unit dosages. Such kits caninclude a card having the dosages oriented in the order of theirintended use. An example of such a kit is a “blister pack”. Blisterpacks are well known in the packaging industry and are widely used forpackaging pharmaceutical unit dosage forms. If desired, a memory aid canbe provided, for example in the form of numbers, letters, or othermarkings or with a calendar insert, designating the days in thetreatment schedule in which the dosages can be administered.

According to one embodiment, a kit may comprise (a) a first containerwith a compound of Formula I contained therein; and optionally (b) asecond container with a second pharmaceutical formulation containedtherein, wherein the second pharmaceutical formulation comprises asecond compound with anti-hyperproliferative activity. Alternatively, oradditionally, the kit may further comprise a third container comprisinga pharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

In certain other embodiments wherein the kit comprises a composition ofFormula I and a second therapeutic agent, the kit may comprise acontainer for containing the separate compositions such as a dividedbottle or a divided foil packet, however, the separate compositions mayalso be contained within a single, undivided container. Typically, thekit comprises directions for the administration of the separatecomponents. The kit form is particularly advantageous when the separatecomponents are preferably administered in different dosage forms (e.g.,oral and parenteral), are administered at different dosage intervals, orwhen titration of the individual components of the combination isdesired by the prescribing physician.

Preparation of Formula I Compounds

Compounds of Formula I may be synthesized by synthetic routes thatinclude processes analogous to those well-known in the chemical arts,particularly in light of the description contained herein, and those forother heterocycles described in: Comprehensive Heterocyclic ChemistryII, Editors Katritzky and Rees, Elsevier, 1997, e.g. Volume 3; LiebigsAnnalen der Chemie, (9):1910-16, (1985); Helvetica Chimica Acta,41:1052-60, (1958); Arzneimittel-Forschung, 40(12):1328-31, (1990), eachof which are expressly incorporated by reference. Starting materials aregenerally available from commercial sources such as Aldrich Chemicals(Milwaukee, Wis.) or are readily prepared using methods well known tothose skilled in the art (e.g., prepared by methods generally describedin Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v.1-23, Wiley, N.Y. (1967-2006 ed.), or Beilsteins Handbuch derorganischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin, includingsupplements (also available via the Beilstein online database).

Synthetic chemistry transformations and protecting group methodologies(protection and deprotection) useful in synthesizing Formula I compoundsand necessary reagents and intermediates are known in the art andinclude, for example, those described in R. Larock, ComprehensiveOrganic Transformations, VCH Publishers (1989); T. W. Greene and P. G.M. Wuts, Protective Groups in Organic Synthesis, 3^(rd) Ed., John Wileyand Sons (1999); and L. Paquette, ed., Encyclopedia of Reagents forOrganic Synthesis, John Wiley and Sons (1995) and subsequent editionsthereof.

Compounds of Formula I may be prepared singly or as compound librariescomprising at least 2, for example 5 to 1,000 compounds, or 10 to 100compounds. Libraries of compounds of Formula I may be prepared by acombinatorial ‘split and mix’ approach or by multiple parallel synthesesusing either solution phase or solid phase chemistry, by proceduresknown to those skilled in the art. Thus according to a further aspect ofthe invention there is provided a compound library comprising at least 2compounds, or pharmaceutically acceptable salts thereof.

The Figures and Examples provide exemplary methods for preparing FormulaI compounds. Those skilled in the art will appreciate that othersynthetic routes may be used to synthesize the Formula I compounds.Although specific starting materials and reagents are depicted anddiscussed in the Figures and Examples, other starting materials andreagents can be easily substituted to provide a variety of derivativesand/or reaction conditions. In addition, many of the exemplary compoundsprepared by the described methods can be further modified in light ofthis disclosure using conventional chemistry well known to those skilledin the art.

In preparing compounds of Formulas I, protection of remote functionality(e.g., primary or secondary amine) of intermediates may be necessary.The need for such protection will vary depending on the nature of theremote functionality and the conditions of the preparation methods.Suitable amino-protecting groups include acetyl, trifluoroacetyl,t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz) and9-fluorenylmethyleneoxycarbonyl (Fmoc). The need for such protection isreadily determined by one skilled in the art. For a general descriptionof protecting groups and their use, see T. W. Greene, Protective Groupsin Organic Synthesis, John Wiley & Sons, New York, 1991.

Experimental procedures, intermediates and reagents useful for usefulfor the preparation of Formula I compounds may be found in U.S. Ser. No.13/102,720, “PYRIDONE AND AZA-PYRIDONE COMPOUNDS AND METHODS OF USE”,filed 6 May 2011, which is incorporated by reference in its entirety.

FIGS. 1-14 describe the synthesis of exemplary embodiments of Formula Icompounds 101-115, more fully described in Examples 101-114, and may beuseful for the preparation of other Formula I compounds.

General Preparative Procedures

General Procedure:

Suzuki Coupling

The Suzuki-type coupling reaction is useful to form carbon-carbon bondsto attach the rings of Formula I compounds and intermediates such as A-3(Suzuki (1991) Pure Appl. Chem. 63:419-422; Miyaura and Suzuki (1979)Chem. Reviews 95(7):2457-2483; Suzuki (1999) J. Organometal. Chem.576:147-168). Suzuki coupling is a palladium mediated cross couplingreaction of a heteroarylhalide, such as B-2 or B-4, with a boronic acidsuch as A-1 or A-2. For example, B-2 may be combined with about 1.5equivalents of4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane), anddissolved in about 3 equivalents of sodium carbonate as a 1 molarsolution in water and an equal volume of acetonitrile. A catalyticamount, or more, of a low valent palladium reagent, such asbis(triphenylphosphine)palladium(II) dichloride, is added. In some casespotassium acetate is used in place of sodium carbonate to adjust the pHof the aqueous layer. The reaction is then heated to about 140-150° C.under pressure in a microwave reactor (Biotage AB, Uppsala, Sweden) for10 to 30 minutes. The contents are extracted with ethyl acetate, oranother organic solvent. After evaporation of the organic layer theboron ester A-1 may be purified on silica or by reverse phase HPLC.Substituents are as defined, or protected forms or precursors thereof.Likewise, bromide intermediate B-4 can be boronylated to give A-2.

Suzuki coupling of B-2 and A-2, or of A-1 and B-4, gives Formula Icompound or intermediate A-3. Boronic ester (or acid) (1.5 eq) A-1 orA-2, and a palladium catalyst such asbis(triphenylphosphine)palladium(II) chloride (0.05 eq) is added to amixture of halo intermediate (1 eq) B-2 or B-4 in acetonitrile and 1 Mof sodium carbonate aqueous solution (equal volume as acetonitrile). Thereaction mixture is heated to about 150° C. in a microwave for about 15min. LC/MS indicates when the reaction is complete. Water is added tothe mixture, and the precipitated product is filtered and purified byHPLC to yield the product A-3. Substituents are as defined, or protectedforms or precursors thereof.

A variety of palladium catalysts can be used during the Suzuki couplingstep. Various low valent, Pd(II) and Pd(0) catalysts may be used in theSuzuki coupling reaction, including PdCl2(PPh₃)₂, Pd(t-Bu)₃, PdCl₂ dppfCH₂Cl₂, Pd(PPh₃)₄, Pd(OAc)/PPh₃, Cl₂Pd[(Pet₃)]₂, Pd(DIPHOS)₂,Cl₂Pd(Bipy), [PdCl(Ph₂PCH₂PPh₂)]₂, Cl₂Pd[P(o-tol)₃]₂,Pd₂(dba)₃/P(o-tol)₃, Pd₂(dba)/P(furyl)₃, Cl₂Pd[P(furyl)₃]₂,Cl₂Pd(PMePh₂)₂, Cl₂Pd[P(4-F-Ph)₃]₂, Cl₂Pd[P(C₆F₆)₃]₂,Cl₂Pd[P(2-COOH-Ph)(Ph)₂]₂, Cl₂Pd[P(4-COOH-Ph)(Ph)₂]₂, and encapsulatedcatalysts Pd EnCat™ 30, Pd EnCat™ TPP30, and Pd(II)EnCat™ BINAP30 (US2004/0254066).

General Procedure:

Buchwald Reaction

The Buchwald reaction is useful to aminate 6-bromo intermediates B-1(Wolf and Buchwald (2004) Org. Synth Coll. Vol. 10:423; Paul et al(1994) Jour. Amer. Chem. Soc. 116:5969-5970). To a solution of halointermediate B-1 in DMF is added the appropriate amine R⁵—NH₂ (200 mol%), Cs₂CO₃ (50 mol %), Pd₂(dba)₃ (5 mol %), and4,5-bis(diphenylphosphino)-9,9-dimethylxanthene (Xantphos, CAS Reg. No.161265-03-8, 10 mol %). The reaction is heated to about 110° C. underpressure in a microwave reactor (Biotage AB, Uppsala, Sweden) for about30 min. The resulting solution is concentrated in vacuo to give B-2.Other palladium catalysts and phosphine ligands may be useful.

N-Heteroaryl amide intermediates B-4 can also be prepared under Buchwaldconditions with cyclic amide intermediates (R⁷) such as3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 101g and heteroaryldibromides B-3.

Methods of Separation

In the methods of preparing Formula I compounds, it may be advantageousto separate reaction products from one another and/or from startingmaterials. The desired products of each step or series of steps isseparated and/or purified to the desired degree of homogeneity by thetechniques common in the art. Typically such separations involvemultiphase extraction, crystallization from a solvent or solventmixture, distillation, sublimation, or chromatography. Chromatographycan involve any number of methods including, for example: reverse-phaseand normal phase; size exclusion; ion exchange; high, medium and lowpressure liquid chromatography methods and apparatus; small scaleanalytical; simulated moving bed (SMB) and preparative thin or thicklayer chromatography, as well as techniques of small scale thin layerand flash chromatography.

Another class of separation methods involves treatment of a mixture witha reagent selected to bind to or render otherwise separable a desiredproduct, unreacted starting material, reaction by product, or the like.Such reagents include adsorbents or absorbents such as activated carbon,molecular sieves, ion exchange media, or the like. Alternatively, thereagents can be acids in the case of a basic material, bases in the caseof an acidic material, binding reagents such as antibodies, bindingproteins, selective chelators such as crown ethers, liquid/liquid ionextraction reagents (LIX), or the like. Selection of appropriate methodsof separation depends on the nature of the materials involved, such as,boiling point and molecular weight in distillation and sublimation,presence or absence of polar functional groups in chromatography,stability of materials in acidic and basic media in multiphaseextraction, and the like.

Diastereomeric mixtures can be separated into their individualdiastereomers on the basis of their physical chemical differences bymethods well known to those skilled in the art, such as bychromatography and/or fractional crystallization. Enantiomers can beseparated by converting the enantiomeric mixture into a diastereomericmixture by reaction with an appropriate optically active compound (e.g.,chiral auxiliary such as a chiral alcohol or Mosher's acid chloride),separating the diastereomers and converting (e.g., hydrolyzing) theindividual diastereoisomers to the corresponding pure enantiomers. Also,some of the compounds of the present invention may be atropisomers(e.g., substituted biaryls) and are considered as part of thisinvention. Enantiomers can also be separated by use of a chiral HPLCcolumn.

A single stereoisomer, e.g., an enantiomer, substantially free of itsstereoisomer may be obtained by resolution of the racemic mixture usinga method such as formation of diastereomers using optically activeresolving agents (Eliel, E. and Wilen, S. “Stereochemistry of OrganicCompounds,” John Wiley & Sons, Inc., New York, 1994; Lochmuller, C. H.,(1975) J. Chromatogr., 113(3):283-302). Racemic mixtures of chiralcompounds of the invention can be separated and isolated by any suitablemethod, including: (1) formation of ionic, diastereomeric salts withchiral compounds and separation by fractional crystallization or othermethods, (2) formation of diastereomeric compounds with chiralderivatizing reagents, separation of the diastereomers, and conversionto the pure stereoisomers, and (3) separation of the substantially pureor enriched stereoisomers directly under chiral conditions. See: “DrugStereochemistry, Analytical Methods and Pharmacology,” Irving W. Wainer,Ed., Marcel Dekker, Inc., New York (1993).

Under method (1), diastereomeric salts can be formed by reaction ofenantiomerically pure chiral bases such as brucine, quinine, ephedrine,strychnine, α-methyl-β-phenylethylamine (amphetamine), and the like withasymmetric compounds bearing acidic functionality, such as carboxylicacid and sulfonic acid. The diastereomeric salts may be induced toseparate by fractional crystallization or ionic chromatography. Forseparation of the optical isomers of amino compounds, addition of chiralcarboxylic or sulfonic acids, such as camphorsulfonic acid, tartaricacid, mandelic acid, or lactic acid can result in formation of thediastereomeric salts.

Alternatively, by method (2), the substrate to be resolved is reactedwith one enantiomer of a chiral compound to form a diastereomeric pair(E. and Wilen, S. “Stereochemistry of Organic Compounds”, John Wiley &Sons, Inc., 1994, p. 322). Diastereomeric compounds can be formed byreacting asymmetric compounds with enantiomerically pure chiralderivatizing reagents, such as menthyl derivatives, followed byseparation of the diastereomers and hydrolysis to yield the pure orenriched enantiomer. A method of determining optical purity involvesmaking chiral esters, such as a menthyl ester, e.g., (−) menthylchloroformate in the presence of base, or Mosher ester,α-methoxy-α-(trifluoromethyl)phenyl acetate (Jacob III. J. Org. Chem.(1982) 47:4165), of the racemic mixture, and analyzing the ¹H NMRspectrum for the presence of the two atropisomeric enantiomers ordiastereomers. Stable diastereomers of atropisomeric compounds can beseparated and isolated by normal- and reverse-phase chromatographyfollowing methods for separation of atropisomeric naphthyl-isoquinolines(WO 96/15111). By method (3), a racemic mixture of two enantiomers canbe separated by chromatography using a chiral stationary phase (“ChiralLiquid Chromatography” (1989) W. J. Lough, Ed., Chapman and Hall, NewYork; Okamoto, J. Chromatogr., (1990) 513:375-378). Enriched or purifiedenantiomers can be distinguished by methods used to distinguish otherchiral molecules with asymmetric carbon atoms, such as optical rotationand circular dichroism.

EXAMPLES Example 101a 6-Chloro-8-bromoimidazo[1,2-a]pyridine 101a

A mixture of 3-bromo-5-chloropyridin-2-amine (10 g, 49 mmol) andchloroacetaldehyde (50% in H₂O, 12 mL, 98 mmol) in ethanol (100 mL) washeated at 50° C. overnight. It was then cooled to room temperature andconcentrated. Acetone (30 mL) was added to the residue and the resultingmixture was stirred rapidly for 2 h. The resulting solid was collectedthrough filtration and dried to afford 101a as a yellow solid (10.0 g,89%). MS: [M+H]⁺ 231. ¹H NMR (500 MHz, DMSO) δ 9.20 (s, 1H), 8.33 (s,1H), 8.29 (s, 1H), 8.09 (s, 1H)

Example 101b6-Chloro-N-(5-(4-methylpiperazin-1-yl)pyridin-2-yl)imidazo[1,2-a]pyridin-8-amine101b

A mixture of 101a (2.3 g, 10 mmol),5-(4-methylpiperazin-1-yl)pyridin-2-amine (1.9 g, 10 mmol), XantPhos(576 mg, 1.0 mmol), Pd₂(dba)₃ (915 mg, 1.0 mmol) and Cs₂CO₃ (6.5 g, 20mmol) in dioxane (50 mL) was heated at 100° C. for 12 h under nitrogen.It was then filtered and evaporated in vacuo. The residue was purifiedby silica-gel column eluting with 1:1 ethyl acetate/petroleum ether toafford 101b as a green solid (1.5 g, 44%). MS: (M+H)⁺ 343.

Example 101c2,2,2-Trichloro-1-(4,5,6,7-tetrahydro-1H-indol-2-yl)ethanone 101c

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer, condenser and nitrogen inlet was purged with nitrogen andcharged with 4,5,6,7-tetrahydro-1H-indole (3.00 g, 24.8 mmol),trichloroacetyl chloride (13.5 g, 74.4 mmol) and 1,2-dichloroethane (50mL). The solution was stirred at 85° C. for 2 h. After that time, thereaction mixture was concentrated under reduced pressure to afford a100% yield (6.50 g) of 101c as a black semi-solid: ¹H NMR (500 MHz,DMSO-d₆) δ 11.94 (s, 1H), 7.05 (s, 1H), 2.62 (t, 2H, J=6.0 Hz), 2.47 (t,2H, J=6.0 Hz), 1.80 (m, 2H), 1.65 (m, 2H); MS (ESI+) m/z 266.0 (M+H)

Example 101d Ethyl 4,5,6,7-Tetrahydro-1H-indole-2-carboxylate 101d

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and nitrogen inlet was purged with nitrogen and charged with101c (6.50 g, 24.8 mmol), sodium ethoxide (17.0 mg, 0.25 mmol) andethanol (40 mL). The solution was stirred at room temperature for 1 h.After that time, the reaction mixture was concentrated under reducedpressure. The residue was purified by column chromatography to afford a100% yield (4.80 g) of 101d as a brown solid: mp 70-72° C.; ¹H NMR (300MHz, CDCl₃) δ 9.08 (s, 1H), 6.75 (s, 1H), 4.25 (q, 2H, J=7.2 Hz), 2.65(t, 2H, J=6.0 Hz), 2.56 (t, 2H, J=6.0 Hz), 1.85 (m, 4H), 1.28 (t, 3H,J=7.2 Hz); MS (ESI+) m/z 194.1 (M+H)

Example 101e Ethyl1-(Cyanomethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxylate 101e

A 125-mL single-neck round-bottomed flask equipped with a magneticstirrer and nitrogen inlet was purged with nitrogen and charged with101d (5.76 g, 29.8 mmol) and DMF (50 mL). The solution was cooled to 0°C. using an ice bath. NaH (60% dispersion in mineral oil, 1.43 g, 35.8mmol) was added. The resulting mixture was stirred at room temperaturefor 1 h. After that time, bromoacetonitrile (1.43 g, 35.8 mmol) wasadded. The mixture was stirred at room temperature for 14 h. After thattime, the reaction mixture was concentrated under reduced pressure andthe residue was partitioned between ethyl acetate (150 mL) and water(450 mL). The organic layer was separated, and the aqueous layer wasextracted with ethyl acetate (3×150 mL). The combined organic layerswere washed with brine, dried over sodium sulfate and concentrated underreduced pressure. The residue was purified by column chromatography toafford a 55% yield (3.80 g) of 101e as a yellow semi-solid: ¹H NMR (300MHz, CDCl₃) δ 6.66 (s, 1H), 5.29 (s, 2H), 4.28 (q, 2H, J=7.2 Hz), 2.62(t, 2H, J=6.3 Hz), 2.49 (t, 2H, J=6.3 Hz), 1.92 (m, 2H), 1.75 (m, 2H),1.33 (t, 3H, J=7.2 Hz); MS (ESI+) m/z 233.1 (M+H)

Example 101f Ethyl1-(2-Aminoethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxylate 101f

A 200-mL Parr reactor bottle was purged with nitrogen and charged with10% palladium on carbon (50% wet, 1.28 g dry weight), 101e (3.00 g, 12.9mmol), 12% hydrochloric acid (6.5 mL, 25 mmol), ethyl acetate (60 mL)and ethanol (40 mL). The bottle was attached to a Parr hydrogenator,evacuated, charged with hydrogen gas to a pressure of 50 psi and shakenfor 6 h. After this time, the hydrogen was evacuated, and nitrogen wascharged into the bottle. Diatomaceous earth filtration agent (Celite®521, 4.0 g) was added, and the mixture was filtered through a pad ofCelite 521. The filter cake was washed with ethanol (2×20 mL), and thecombined filtrates were concentrated to dryness under reduced pressure.The residue was partitioned between ethyl acetate (150 mL) and 10%aqueous potassium carbonate (100 mL). The organic layer was separated,and the aqueous layer was extracted with ethyl acetate (3×75 mL). Thecombined organic layers were dried over sodium sulfate and concentratedunder reduced pressure. The residue was triturated with ethanol (5 mL)to afford a 71% yield (1.71 g) of ethyl1-(2-aminoethyl)-4,5,6,7-tetrahydro-1H-indole-2-carboxylate 101f as awhite solid: mp 102-104° C.; ¹H NMR (500 MHz, DMSO-d₆) δ 6.61 (s, 1H),6.22 (br, 2H), 4.15 (m, 4H), 2.77 (m, 2H), 2.59 (t, 2H, J=6.5 Hz), 2.42(t, 2H, J=6.5 Hz), 1.70 (m, 2H), 1.62 (m, 2H), 1.23 (t, 3H, J=7.0 Hz);MS (APCI+) m/z 237.2 (M+H)

Example 101g 3,4,6,7,8,9-Hexahydropyrazino[1,2-a]indol-1(2H)-one 101g

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and nitrogen inlet was purged with nitrogen and charged with101f (1.80 g, 7.63 mmol), sodium ethoxide (1.55 g, 22.8 mmol) andethanol (50 mL). The mixture was stirred at 55° C. for 5 h. After thattime, the reaction mixture was concentrated under reduced pressure andthe residue was partitioned between ethyl acetate (200 mL) and water(100 mL). The organic layer was separated, and the aqueous layer wasextracted with ethyl acetate (2×100 mL). The combined organic layerswere washed with brine, dried over sodium sulfate and concentrated underreduced pressure. The residue was purified by column chromatography toafford a 42% yield (605 mg) of 101g as a white solid: mp 207-209° C.; ¹HNMR (500 MHz, DMSO-d₆) δ 7.41 (s, 1H), 6.36 (s, 1H), 3.84 (t, 2H, J=6.0Hz), 3.42 (m, 2H), 2.51 (t, 2H, J=6.0 Hz), 2.42 (t, 2H, J=6.0 Hz), 1.76(m, 2H), 1.65 (m, 2H); (APCI+) m/z 191.3 (M+H)

Example 101h 2,6-Dibromo-4-fluorobenzaldehyde 101h

To a solution of 1,3-dibromo-5-fluoro-2-iodobenzene (50 g, 132 mmol) inanhydrous toluene (300 mL) cooled at −35° C. was added the solution ofisopropylmagnesium chloride (84 mL, 171 mmol, 2.0M in Et₂O) over 30minutes while maintaining the internal temperature below −25° C. A clearbrown solution was obtained and the stirring was continued for 1.5 h at−25° C. Then anhydrous DMF (34 mL, 436 mmol) was added over a period of30 minutes. The reaction mixture was warmed to 10° C. (room temperature)over 1 h and stirred at this temperature for 1.5 h. It was then quenchedwith 3.0M HCl and followed by the addition of ethyl acetate. The organiclayer was separated and evaporated under reduced pressure. The residuewas purified by silica-gel column chromatography eluting with petroleumether/ethyl acetate (from 50:1 to 20:1) to give 101h as a white solid(20 g, 54%).

¹H NMR (500 MHz, CDCl₃) δ 10.23 (s, 1H), 7.48 (d, J=7.5, 2H).

Example 101i (2,6-Dibromo-4-fluorophenyl)methanol 101i

To a solution of 101h (20 g, 71 mmol) in EtOH (500 mL) was added NaBH₄(10 g, 284 mmol). The mixture was stirred at room temperature (10° C.)for 4 h and TLC showed the start material disappeared. The reaction wasquenched by aqueous HCl solution (150 mL, 1M) and evaporated in vacuountil most of EtOH was distilled. The residue was extracted by ethylacetate (500 mL×3). The organic layers were combined, dried with Na₂SO₄,and evaporated in vacuo. The residue was purified by silica-gel columnchromatography eluting with petroleum ether/ethyl acetate (from 50:1 to20:1) to give 101i as a white solid (15 g, 75%). MS: [M−OH]⁺ 267. ¹H NMR(500 MHz, DMSO-d₆) δ 7.68 (d, J=8.5, 2H), 5.23 (s, 1H), 4.71 (s, 2H).

Example 101j 2,6-Dibromo-4-fluorobenzyl Acetate 101j

To a solution of 101i (20 g, 71 mmol) in CH₂Cl₂ (500 mL) at 0° C. wasadded pyridine (8.4 g, 107 mmol) and acetyl chloride (8.3 g, 107 mmol).The mixture was stirred at room temperature for 5 h. TLC showed thestart material disappeared. The reaction was evaporated in vacuum andthe residue was purified by silica-gel column chromatography elutingwith petroleum ether/ethyl acetate (from 50:1 to 20:1) to give 101j as awhite solid (20 g, 87%). MS: [M−Oac]⁺ 267. ¹H NMR (500 MHz, CDCl₃) δ7.36 (d, J=7.5, 2H), 5.38 (s, 2H), 2.10 (s, 3H).

Example 101k2-Bromo-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzylacetate 101k

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer was charged with 101g (3.8 g, 20 mmol), 101j (20 g, 60 mmol),XantPhos (1.2 g, 2 mmol), tris(dibenzylideneacetone)dipalladium(0) (1.8g, 2 mmol), Cs₂CO₃ (16 g, 50 mmol), and 1,4-dioxane (120 mL). The systemwas evacuated and then refilled with N₂. A reflux condenser was attachedto the flask, and the reaction mixture was heated at 100° C. for 16 h.Then, the mixture was cooled to room temperature and filtered. Thefiltrate was concentrated under reduced pressure and the resultingresidue was purified by flash column chromatography eluting with 5:1petroleum ether/ethyl acetate to afford 101k as a white solid (5.2 g,60%). MS: [M+H]⁺ 435. ¹H NMR (500 MHz, DMSO-d₆) δ 7.70 (dd, J=3, 1H),7.48 (dd, J=3, 1H), 6.52 (s, 1H), 5.01 (m, 2H), 4.18 (m, 2H), 4.02 (m,1H), 3.73 (m, 1H), 2.60 (m, 2H), 2.45 (m, 2H), 1.98 (s, 3H), 1.77 (m,2H), 1.68 (m, 2H).

Example 101l4-Fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylacetate 101l

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer was charged with 101k (3.8 g, 8.65 mmol), (PinB)₂ (11 g, 43.25mmol), Pd(dppf)Cl₂ (0.4 g, 0.5 mmol), KOAc (2.5 g, 26 mmol), and1,4-dioxane (150 mL). The system was evacuated and then refilled withN₂. A reflux condenser was attached to the flask and the reactionmixture was heated at 100° C. for 15 h. Then, the mixture was cooled toroom temperature and filtered. The filtrate was concentrated underreduced pressure and the resulting residue was purified by flash columnchromatography eluting with 5:1 petroleum ether/ethyl acetate to afford101l as a yellow solid (3.2 g, 77%). MS: [M+H]⁺ 483.

Example 1012-(5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-a]pyridin-6-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one101

A 25 mL sealed tube was charged with 101b (300 mg, 0.88 mmol), 101l (423mg, 0.88 mmol), Cs₂CO₃ (572 mg, 1.76 mmol), Pd₂(dba)₃ (80 mg, 0.09 mmol)suspended in CH₃CN (25 mL), and H₂O (1 mL). The mixture was heated at140° C. under microwave irradiation for 1 hour. It was then evaporatedand the residue was purified by silica-gel column eluting with 10:1methylene chloride/methanol to give the crude product, which was furtherpurified by reverse phase Combi-flash eluting with 0.3% NH₄HCO₃ in 1:4water/CH₃CN to afford 101 as a yellow solid (150 mg, 28%). MS: (M+H)⁺621. ¹H NMR (500 MHz, DMSO) δ 8.94 (s, 1H), 8.27 (d, J=1.5, 1H), 8.16(d, J=1.5, 1H), 7.96 (d, J=1.0, 1H), 7.91 (d, J=2.5, 1H), 7.58 (d,J=1.5, 1H), 7.33-7.42 (m, 3H), 7.25-7.28 (m, 1H), 6.53 (s, 1H), 4.88 (t,J=4.5, 1H), 4.30 (d, J=4.5, 2H), 4.12-4.20 (m, 3H), 3.90-3.92 (m, 1H),3.06 (t, J=4.5, 4H), 2.53-2.65 (m, 2H), 2.43-2.48 (m, 6H), 2.21 (s, 3H),1.76-1.81 (m, 2H), 1.67-1.71 (m, 2H)

Example 102a tert-Butyl4-(6-(6-chloroimidazo[1,2-a]pyridin-8-ylamino)pyridine-3-yl)piperazine-1-carboxylate102a

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer and reflux condenser was charged with 1,4-dioxane (60 mL),tert-butyl 4-(6-aminopyridin-3-yl)piperazine-1-carboxylate (1.5 g, 5.39mmol), 8-bromo-6-chloroimidazo[1,2-a]pyridine 101a (3.7 g, 16.18 mmol),and cesium carbonate (3.52 g, 10.79 mmol). XantPhos (312 mg, 0.539 mmol)and Pd₂(dba)₃ (494 mg, 0.539 mmol) were added, and the reaction mixturewas heated at 100° C. for 5 h under nitrogen. After this time thereaction was cooled to room temperature. The mixture was filtered andthe filtrate was concentrated under reduced pressure. The residue waspurified on flash column eluting with 100:1 CH₂Cl₂/MeOH to afford 102a(1.8 g, 78%). MS: [M+H]⁺ 429.

Example 102b6-Chloro-N-(5-(piperazin-1-yl)pyridine-2-yl)imidazo[1,2-a]pyridin-8-amine102b

Compound 102a (1.75 g, 4.08 mmol) was suspended in 4.0 M HCl/dioxane (10mL) and stirred at room temperature for 5 h. It was then concentratedunder reduced pressure to afford 102b (1.2 g, 81%). MS: [M+H]⁺ 329.

Example 102c6-Chloro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)imidazo[1,2-a]pyridine-8-amine102c

A mixture of 102b (0.773 g, 1.75 mmol), oxetan-3-one (0.189 g, 2.62mmol), NaBH₃CN (0.22 g, 3.5 mmol), and zinc chloride/Et₂O (3.5 ml, 3.5mmol) in methanol (40 mL) was stirred at 50˜60° C. for 5 hours. Thesolid was removed by filtration and the filtrate was concentrated underreduced pressure. The residue was purified by column chromatographyeluting with 5:1 CH₂Cl₂/methanol to afford 102c (200 mg, 30%). MS:[M+H]⁺ 385.

Example 1022-(5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazine-1-yl)pyridine-2-ylamino)imidazo[1,2-a]pyridin-6-yl)phenyl)-3,4,6,7,8,9-hexahydro-pyrazino[1,2-a]indol-1(2H)-one102

To a solution of 102c (180 mg, 0.468 mol) in dioxane/H₂O (12 mL/1 mL)was added2-(5-fluoro-2-(hydroxymethyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one101l (225 mg, 0.468 mmol), Pd₂(dba)₃ (43 mg, 0.0468 mmol),tricyclohexylphosphine (131 mg, 0.468 mmol), and Cs₂CO₃ (305 mg, 0.935mmol). This mixture was heated in microwave at 140° C. for 1 h. Then,the solid was filtered and the filtrate was concentrated to give ayellow solid, which was further purified by reverse-phase prep-HPLC toafford 102 as a white solid (36 mg, 11%). LCMS: [M+H]⁺ 663. ¹H NMR (500MHz, DMSO) δ 8.53 (d, J=2.5, 1H), 8.27 (s, 1H), 8.16 (s, 1H), 7.95 (s,1H), 7.92-7.91 (d, J=2.5, 1H), 7.57 (s, 1H), 7.42-7.36 (m, 2H),7.36-7.33 (m, 1H), 7.27-7.25 (d, J=9, 1H), 6.53 (s, 1H), 4.88-4.86 (t,J=9, 1H), 4.57-4.54 (m, 2H), 4.47-4.45 (m, 2H), 4.31-4.30 (d, J=4.5,2H), 4.18-4.13 (m, 3H), 3.92-3.90 (m, 1H), 3.45-3.42 (m, 1H), 3.09-3.10(m, 4H), 2.64-2.54 (m, 2H), 2.47-2.45 (m, 2H), 2.39-2.38 (m, 4H),1.79-1.78 (m, 2H), 1.69-1.67 (m, 2H)

Example 103aN-Methoxy-N-methyl-4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxamide103a

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer was purged with nitrogen, charged with4,5,6,7-tetrahydrobenzo[b]thiophene-2-carboxylic acid (3.00 g, 16.5mmol), methylene chloride (80 mL), and DMF (60 mg, 0.825 mmol) andcooled to 0° C. To the resulting solution, oxalyl chloride (2.31 g, 18.2mmol) was added dropwise. After this addition was complete, the reactionwas warmed to room temperature and stirred for 2 h. After this time, thereaction was concentrated to dryness under reduced pressure. Theresulting white solid was dissolved in methylene chloride (80 mL) andthe solution cooled to 0° C. Triethylamine (5.00 g, 49.5 mmol) andN,O-dimethylhydroxylamine (1.61 g, 16.5 mmol) were then added. After theaddition was complete, the cooling bath was removed, and the reactionmixture was stirred at room temperature for 16 h. After this time, thereaction mixture was partitioned between water (100 mL) and ethylacetate (200 mL). The layers were separated, and the aqueous phase wasextracted with ethyl acetate (100 mL). The combined organic extractswere washed with water (100 mL), followed by brine (100 mL) and driedover sodium sulfate. The drying agent was removed by filtration, and thesolvent was evaporated under reduced pressure. The resulting residue waspurified by flash chromatography to afford an 88% yield of 103a (3.29 g)as a white solid: mp 36-37° C.; ¹H NMR (500 MHz, CDCl₃) δ 7.79 (s, 1H),3.76 (s, 3H), 3.34 (s, 3H), 2.78 (t, 2H, J=6.0 Hz), 2.62 (t, 2H, J=6.0Hz), 1.82 (m, 4H); MS (APCI+) m/z 226.3 (M+H)

Example 103b3-Chloro-1-(4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)propan-1-one 103b

A 100-mL single-necked round-bottomed flask equipped with a magneticstirrer was purged with nitrogen and charged with 103a (2.70 g, 12.0mmol) and anhydrous THF (45 mL), and the solution was cooled to −10° C.with acetone/ice bath. A 1.0 M solution of vinylmagnesium bromide in THF(13.2 mL, 13.2 mmol) was added dropwise, and the resulting reactionmixture was stirred at 0° C. for 4 h. After this time, the reactionmixture was partitioned between ethyl acetate (100 mL) and 2 M aqueoushydrochloric acid (40 mL). The layers were separated, and the aqueousphase was extracted with ethyl acetate (40 mL). The combined organicextracts were washed with water (100 mL), followed by brine (100 mL),dried over sodium sulfate, filtered and concentrated under reducedpressure. The resulting residue was dissolved in methylene chloride (30mL), and a 2 M solution of hydrogen chloride in diethyl ether (15 mL)was added. After stirring at room temperature for 1 h, the solvents wereremoved under reduced pressure. Purification of the resulting residue bycolumn chromatography afforded a 29% yield (804 mg) of 103b as anoff-white solid: mp 57-58° C.; ¹H NMR (500 MHz, CDCl₃) δ 7.41 (s, 1H),3.89 (t, 2H, J=7.0 Hz), 3.30 (t, 2H, J=7.0 Hz), 2.81 (t, 2H, J=6.0 Hz),2.64 (t, 2H, J=6.0 Hz), 1.83 (m, 4H); MS (ECI+) m/z 229.1 (M+H)

Example 103c5,6,7,8-Tetrahydro-1H-benzo[b]cyclopenta[d]thiophen-3(2H)-one 103c

A 50-mL single-necked round-bottomed flask equipped with a magneticstirrer was charged with 103b (800 mg, 3.51 mmol) and 98% sulfuric acid(8 mL). After stirring at 95° C. for 16 h, the reaction mixture waspoured into ice (50 g), and the resulting suspension was extracted withethyl acetate (3×50 mL). The organic extracts were combined, dried oversodium sulfate, filtered and concentrated under reduced pressure. Theresulting residue was purified by flash chromatography to afford 103c in47% yield (320 mg) as an off-white solid: mp 75-76° C.; ¹H NMR (500 MHz,CDCl₃) δ 2.89 (m, 2H), 2.87-2.83 (m, 4H), 2.56 (t, 2H, J=6.5 Hz), 1.84(m, 4H)

Example 103d5,6,7,8-Tetrahydro-1H-benzo[b]cyclopenta[d]thiophen-3(2H)-one oxime 103d

A 100-mL single-neck round-bottomed flask equipped with a mechanicalstirrer and nitrogen inlet was charged with hydroxylamine hydrochloride(573 mg, 8.25 mmol) and methanol (10 mL). The mixture was cooled to 0°C. using an ice bath. Sodium acetate (677 mg, 8.25 mmol) was added. Themixture was stirred at 0° C. for 30 min. After this time, 103c (319 mg,1.65 mmol) was added, and the reaction was stirred at room temperaturefor 16 h. After this time, the mixture was concentrated, and theresulting residue was triturated with water (10 mL). The resulting solidwas collected and dried in a vacuum oven at 45° C. to afford an 84%yield (287 mg) of 103d as an off-white solid: mp 173-174° C.; ¹H NMR(500 MHz, DMSO-d₆) δ 10.38 (s, 1H), 2.97 (m, 2H), 2.77-2.73 (m, 4H),2.47 (m, 2H), 1.75 (m, 4H); MS (APCI+) m/z 208.3 (M+H)

Example 103e 3,4,5,6,7,8-Hexahydrobenzothieno[2,3-c]pyridin-1(2H)one103e

A 50-mL single-neck round-bottomed flask equipped with a refluxcondenser, magnetic stirrer and nitrogen inlet was charged with 103d(285 mg, 1.38 mmol) and polyphosphoric acid (15 g). After stirring at80° C. for 16 h, the reaction mixture was cooled to room temperature,and water (30 mL) was added. The resulting mixture was stirred for 30min and filtered. The filter cake was washed with water (20 mL) anddried in a vacuum oven at 45° C. to afford a 75% yield (215 mg) of 103eas an off-white solid: mp 203° C. dec; ¹H NMR (500 MHz, CDCl₃) δ 5.62(s, 1H), 3.59 (t, 2H, J=7.0 Hz), 2.81 (t, 2H, J=6.0 Hz), 2.72 (t, 2H,J=7.0 Hz), 2.48 (t, 2H, J=6.0 Hz), 1.84 (m, 4H). MS (APCI+) m/z 208.3(M+H)

Example 103f2-Bromo-4-fluoro-6-(1-oxo-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-2(1H)-yl)benzylAcetate 103f

A solution of 103e (3 g, 14.5 mmol), 2,6-dibromo-4-fluorobenzyl acetate101j (14 g, 43.5 mmol), Xantphos (839 mg, 1.45 mmol), Pd₂(dba)₃ (1.33 g,1.45 mmol) and Cs₂CO₃ (9.4 g, 29 mmol) in dioxane (200 mL) was heated at100° for 15 h under nitrogen. After filtration, the filtrate wasevaporated in vacuo and purified by flash column eluting with ethylacetate/petroleum ether (1:1) to give 103f (5 g, yield 77%) as a yellowsolid. LCMS: (M+H)⁺452. ¹H NMR (500 MHz, DMSO) δ 7.71 (dd, J=2.5, 1H),7.51 (dd, J=3, 1H), 5.04 (m, 1H), 4.10 (m, 1H), 3.68 (m, 1H), 2.86 (m,2H), 2.77 (m, 2H), 2.55 (m, 3H), 1.98 (s, 3H), 1.78 (m, 4H)

Example 103g2-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-4-fluoro-6-(1-oxo-3,4,5,6,7,8-hexahydrobenzothieno[2,3-c]pyridin-2(1H)-yl)benzylAcetate 103g

A solution of 103f (3 g, 6.65 mmol),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bis(1,3,2-dioxaborolane) (10 g, 40mmol) in dioxane (160 mL) was added PdCl₂(dppf) (543 mg, 0.66 mmol) andCH₃COOK (3.9 g, 40 mmol). The mixture was stirred at 100° for 15 h underargon atmosphere. The mixture was filtered and evaporated in vacuo andpurified by flash column eluting with ethyl acetate/petroleum ether(1:2) to give 103g (2.5 g, yield 76%) as a yellow solid. LCMS: (M+H)⁺500

Example 1035-[5-Fluoro-2-(hydroxymethyl)-3-{(8-(5-(4-methylpiperazin-1-yl)pyridine-2-ylamino)imidazo[1,2-a]pyridin-6-yl)}phenyl]-8-thia-5-azatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7)-dien-6-one103

Following the procedures as described for compound 101, 103g (499 mg,1.0 mmol), and6-chloro-N-(5-(4-methylpiperazin-1-yl)pyridin-2-yl)imidazo[1,2-a]pyridin-8-amine101b (342 mg, 1.0 mmol) were reacted to give 103 as a white solid (220mg, 35%). LCMS: [M+H]⁺ 638. ¹H NMR (500 MHz, CDCl₃) δ 8.22 (s, 2H), 7.96(d, J=2.5, 1H), 7.83 (s, 1H), 7.63 (d, J=1.0, 1H), 7.57 (d, J=1.0, 1H),7.30 (dd, J=3.0, 9.0, 1H), 7.24 (dd, J=2.5, 9.0, 1H), 7.03 (dd, J=3.0,9.0, 1H), 6.92 (d, J=8.5, 1H), 4.57 (d, J=11.5, 1H), 4.35 (t, J=8.5,1H), 4.21 (s, 1H), 4.09-4.15 (m, 1H), 3.87-3.92 (m, 1H), 3.16 (t, J=5.0,4H), 2.85-3.02 (m, 4H), 2.60 (t, J=5.0, 4H), 2.51-2.57 (m, 2H), 2.37 (s,3H), 1.85-1.95 (m, 4H)

Example 104a 8-Bromo-6-chloroimidazo[1,2-b]pyridazine 104a

A solution of 4-bromo-6-chloropyridazin-3-amine (5.0 g, 24 mmol) and2-chloroacetaldehyde (12 g, 75 mmol) in ethanol (100 mL) was heated atreflux for 15 h. The reaction mixture was concentrated and washed withacetone (75 mL) to give 104a as a yellow solid (6.0 g, 71%). MS: [M+H]⁺234.

Example 104b6-Chloro-N-(5-(4-methylpiperazin-1-yl)pyridin-2-yl)imidazo[1,2-b]pyridazin-8-amine104b

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 104a (2.0 g, 4 mmol),5-(4-methylpiperazin-1-yl)pyridin-2-amine (920 mg, 4.8 mmol),tris(dibenzylideneacetone)dipalladium(0) (366 mg, 0.4 mmol), XantPhos(688 mg, 1.2 mmol), Cs₂CO₃ (2.6 g, 8.0 mmol), and 1,4-dioxane (60 mL).After three cycles of vacuum/argon flush, the mixture was heated atreflux for 15 h. It was then cooled to room temperature and filtered.The filtrate was concentrated under reduced pressure and the resultingresidue was purified by silica-gel column chromatography eluting withdichloromethane/methanol (20:1) to afford 104b as yellow solid (1.4 g,70%). MS: [M+H]⁺344.

Example 1042-(5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one104

A sealed tube was charged with 104b (340 mg, 0.8 mmol),4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylacetate 101l (400 mg, 0.8 mmol), Pd₂(dba)₃ (72 mg, 0.1 eq., 0.08 mmol),PCy₃ (68 mg, 0.3 eq., 0.24 mmol), cesium carbonate (520 mg, 2 eq., 1.6mmol), dioxane (15 mL), and water (1 mL). After three cycles ofvacuum/argon flash, the mixture was heated at 130° C. for 14 h. It wasthen cooled to room temperature and filtered. The filtrate wasconcentrated under reduced pressure and the resulting residue waspurified by silica-gel column chromatography eluting withdichloromethane/-methanol (20:1) to afford 104 (100 mg, 16%). LCMS:[M+H]⁺ 622. ¹H NMR (500 MHz, DMSO) δ 9.95 (s, 1H), 8.23 (s, 1H), 8.16(s, 1H), 8.00 (d, J=2.0, 1H), 7.67 (s, 1H), 7.43-7.46 (m, 3H), 7.34-7.36(m, 1H), 6.52 (s, 1H), 4.67 (t, J=5.0, 1H), 4.34-4.41 (m, 2H), 4.13-4.18(m, 3H), 3.87-3.88 (m, 1H), 3.10-3.12 (m, 4H), 2.57-2.61 (m, 2H),2.43-2.47 (m, 6H), 2.21 (s, 3H), 1.77-1.79 (m, 2H), 1.68-1.69 (m, 2H)

Example 105a6-Chloro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)imidazo[1,2-b]pyridazin-8-amine105a

Following the procedures as described for compound 104b,8-bromo-6-chloroimidazo[1,2-b]pyridazine 104a (233 mg, 1.0 mmol), and5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-amine (234 mg, 1.0 mmol) werereacted to give 105a as a white solid (296 mg, 77%). LCMS: [M+H]⁺ 386

Example 1052-(5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)phenyl)-3,4,6,7,8,9-hexahydro-pyrazino[1,2-a]indol-1(2H)-one105

Following the procedures as described for compound 104, Suzuki reactionof 105a (385 mg, 1.0 mmol) and4-Fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylacetate 101l gave 105 as a yellow solid (60 mg, 9%). LCMS: [M+H]⁺ 664.¹H NMR (500 MHz, DMSO) δ 10.04 (d, J=4.0, 1H), 8.26 (s, 1H), 8.18 (s,1H), 8.10-8.13 (m, 1H), 7.70 (s, 1H), 7.44-7.55 (m, 3H), 7.34-7.36 (m,1H), 6.52 (s, 1H), 4.84 (s, 1H), 4.66-4.72 (m, 3H), 4.33-4.43 (m, 2H),4.11-4.19 (m, 3H), 3.86-3.89 (m, 2H), 3.49-3.51 (m, 2H), 3.03-3.14 (m,4H), 2.57-2.63 (m, 3H), 2.45-2.47 (m, 4H), 1.77-1.80 (m, 2H), 1.68-1.69(m, 2H)

Example 106a tert-Butyl4-(6-(6-Bromoimidazo[1,2-a]pyrazin-8-ylamino)pyridin-3-yl)piperazine-1-carboxylate106a

A mixture of 6,8-dibromoimidazo[1,2-a]pyrazine (1.2 g, 4.3 mmol),diisopropylethylamine (1.1 g, 8 mmol), and tert-butyl4-(6-aminopyridin-3-yl)piperazine-1-carboxylate (1 g, 3.5 mmol) in IPA(20 mL) was stirred at 130° C. under microwave irradiation for 1.5 h.The resulting suspension was filtered and the solid was washed withwater and dried in vacuum to afford the crude product, which was furtherpurified by silica gel chromatography eluting with CH₂Cl₂/MeOH (50:1) toafford 106a as a white solid (800 mg, 50%). MS: [M+H]⁺ 474.

Example 106b6-Bromo-N-(5-(piperazin-1-yl)pyridin-2-yl)imidazo[1,2-a]pyrazin-8-amine106b

To a mixture of 106a (260 mg, 0.5 mmol) in CH₂Cl₂ (3 mL) was added TFA(0.5 ml) and the reaction mixture was stirred at 25° C. for 2 h. Thereaction solution was concentrated to give 106b as a pale yellow liquidwithout purification for next step (210 mg, 97%). MS: [M+H]⁺ 374.

Example 106c6-Bromo-N-(5-(4-methylpiperazin-1-yl)pyridin-2-yl)imidazo[1,2-a]pyrazin-8-amine106c

A mixture of 106b (210 mg, 0.52 mmol), formaldehyde (150 mg, 5 mmol),and NaBH₃CN (220 mg, 3.5 mmol) in methanol (8 mL) was stirred at 20° C.for 3 hours. The solid was removed by filtration and the filtrate wasconcentrated under reduced pressure. The residue was purified bycolumn-chromatography eluting with CH₂Cl₂/methanol (20:1) to afford 106c(200 mg, 90%). MS: [M+H]⁺ 388.

Example 106d4-Fluoro-2-(8-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)imidazo-[1,2-a]pyrazin-6-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzylAcetate 106d

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer and reflux condenser was charged with 1,4-dioxane (60 mL), 106c(200 mg, 0.5 mmol),4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylacetate 101l (385 mg, 0.8 mmol), and cesium carbonate (332 mg, 1 mmol).Xantphos (30 mg, 0.08 mmol) and Pd₂(dba)₃ (55 mg, 0.08 mmol) were added,and the reaction mixture was heated at 90° C. for 4 h. After this timethe reaction was cooled to room temperature, it was filtered and thefiltrate was concentrated under reduced pressure. The residue waspurified on flash column eluting with CH₂Cl₂/MeOH (10:1) to afford 106d(200 mg, 60%). MS: [M+H]⁺ 664.

Example 1062-(5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-a]pyrazin-6-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one106

A mixture of 106d (200 mg, 0.92 mmol) and LiOH (300 mg, 10 mmol) in^(i)PrOH/THF (1:1, 3.5 mL) and H₂O (1 mL) was stirred at 50° C. for 0.5h. The mixture was evaporated in vacuo and the residue was extractedwith Ethyl acetate (10 mL×2). The combined Ethyl acetate extract wasconcentrated under reduced pressure and the residue was purified withreverse-phase prep-HPLC to afford 106 (45 mg, 20%). LCMS: [M+H]⁺ 622. ¹HNMR (500 MHz, DMSO) δ 9.04 (s, 1H), 8.41 (s, 1H), 8.13-8.10 (m, 2H),7.98 (s, 1H), 7.71 (s, 1H), 7.47-7.44 (m, 2H), 7.39-7.37 (m, 1H), 6.53(s, 1H), 5.38-5.36 (m, 1H), 4.37-4.44 (m, 2H), 4.19-3.98 (m, 4H), 3.12(s, 4H), 2.60-2.58 (m, 2H), 2.50-2.45 (m, 6H), 2.21 (s, 3H), 1.79-1.68(m, 2H), 1.69-1.67 (m, 2H)

Example 107a2-(5-Fluoro-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-imidazo[1,2-a]pyrazin-6-yl)-2-(2-oxopropyl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one107a

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer and reflux condenser was charged with 1,4-dioxane (60 mL),6-bromo-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)imidazo[1,2-a]pyrazin-8-amine(130 mg, 0.3 mmol),4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylacetate 101l (146 mg, 0.3 mmol), PdCl₂(dppf) (25 mg, 0.03 mmol), K₃PO₄(138 mg, 0.6 mmol), and NaOAc (48 mg, 0.6 mmol) in CH₃CN (5 mL) and H₂O(1.5 mL). The system was evacuated and refilled with N₂. The reactionmixture was heated at 100° C. for 2 h. It was then cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure and the resulting residue was purified by flash columnchromatography eluting with 10:1 CH₂Cl₂/MeOH to afford 107a (150 mg,70%) as a brown solid. MS: [M+H]⁺ 706.4.

Example 1072-(5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-a]pyrazin-6-yl)phenyl)-3,4,6,7,8,9-hexahydro-pyrazino[1,2-a]indol-1(2H)-one107

A 100-mL single-neck round-bottomed flask was charged with 107a (150 mg,0.21 mol) in THF/iPA/H₂O (5 mL/5 mL/2 mL) and LiOH (85 mg, 3.5 mmol)while stirring. This mixture was stirred at 50° C. for 0.5 h. Then 20 mLH₂O was added and the mixture was extracted with EA (30 mL×3). Thecombined organic layer was dried over Na₂SO₄ and concentrated to give ayellow solid, which was further purified by reverse-phase prep-HPLC toafford 107 as a white solid (74 mg, 52% yield). LCMS: [M+H]⁺ 664.3. ¹HNMR (500 MHz, DMSO) δ 9.06 (s, 1H), 8.41 (s, 1H), 8.13-8.10 (m, 2H),7.98 (d, J=2.5, 1H), 7.71 (s, 1H), 7.48-7.44 (m, 2H), 7.39-7.36 (m, 1H),6.54 (s, 1H), 5.38 (m, 1H), 4.57-4.35 (m, 6H), 4.18-4.13 (m, 4H),3.44-3.42 (m, 1H), 3.15 (s, 4H), 2.59 (d, J=3.0, 2H), 2.47-2.40 (m, 6H),1.79-1.69 (m, 4H)

Example 108a(E)-N′-(3-Bromo-5-chloropyridin-2-yl)-N,N-dimethylformimidamide 108a

A mixture of 3-bromo-5-chloropyridin-2-amine (10 g, 0.05 mol) anddimethoxy-N,N-dimethylmethanamine (7 g, 0.06 mmol) was stirred at 100°C. for 1 h. The mixture was poured into water and then extracted withEthyl acetate (20 mL×4). The combined organic layer was concentratedunder reduced pressure and the residue was purified with silica gelchromatography eluting with Ethyl acetate/PE (1:2) to afford 108a (10 g,77%). LCMS: [M+H]⁺ 262.

Example 108b(E)-N′-(3-bromo-5-chloropyridin-2-yl)-N-hydroxyformimidamide 108b

A mixture of 108a (2 g, 10 mmol) and hydroxylamine hydrochloride (1.4 g,20 mmol) in MeOH (10 mL) was stirred at 100° C. for 0.5 h. The mixturewas extracted with Ethyl acetate (20 mL×4) and the combined organicphase was washed with water. It was then concentrated under reducedpressure to afford 108b (2 g, 80%), which was used for next step withoutfurther purification. LCMS: [M+H]⁺ 250.

Example 108c 8-Bromo-6-chloro-[1,2,4]triazolo[1,5-a]pyridine 108c

A mixture of 108b (500 mg, 2 mmol) and PPA (1.4 g, 20 mmol) was stirredat 100° C. for 4 h. The mixture was then extracted with Ethyl acetate(20 mL×4) and the combined organic phase was washed with water. It wasconcentrated under reduced pressure to 108c (250 mg, 50%), which wasused for next step without further purification. LCMS: [M+H]⁺ 250.

Example 108d6-Chloro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)-[1,2,4]-triazolo[1,5-a]pyridin-8-amine108d

A 25-mL single-neck round-bottomed flask equipped with a magneticstirrer and reflux condenser was charged with 1,4-dioxane (8 mL), 108c(250 mg, 1.07 mmol), 5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-amine(252 mg, 1 mmol), and cesium carbonate (700 mg, 2 mmol). Xantphos (30mg, 0.08 mmol) and Pd₂(dba)₃ (55 mg, 0.08 mmol) were added, and thereaction mixture was heated at 105° C. for 3 h. After this time thereaction was cooled to room temperature and filtered. The filtrate wasconcentrated under reduced pressure and the residue was purified bysilica gel chromatography eluting with CH₂Cl₂/MeOH (20:1) to afford 108d(250 mg, 60%). MS: [M+H]⁺ 386.

Example 108e4-Fluoro-2-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzylacetate 108e

A mixture of4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylacetate 101l (165 mg, 0.38 mmol), 108d (140 mg, 0.38 mmol), PdCl₂(dppf)(41 mg, 0.056 mmol), K₃PO₄ (100 mg), and NaOAc (50 mg) in MeCN (10 mL)and H₂O (3 mL) was heated at 110° C. in sealed tube for 2 h. The solventwas evaporated in vacuo and the residue was purified by silica gelchromatography to afford 108e (250 mg, 60%). MS: [M+H]⁺ 706.

Example 1082-(5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-[1,2,4]triazolo[1,5-a]pyridin-6-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one108

A mixture of 108e (250 mg, 0.35 mmol) and LiOH (300 mg, 10 mmol) in^(i)PrOH/THF (1:1, 3.5 mL) and H₂O (1 mL) was stirred at 50° C. for 0.5h. The mixture was evaporated in vacuo and the residue was extractedwith Ethyl acetate (5 mL×2). The combined Ethyl acetate extract wasconcentrated under reduced pressure and the residue was purified withreverse-phase prep-HPLC to afford 108 (110 mg, 25%). LCMS: [M+H]⁺ 664.¹H NMR (500 MHz, DMSO) δ 9.33 (s, 1H), 8.67 (s, 1H), 8.55-8.54 (m, 2H),7.95 (d, J=3.0, 1H), 7.44-7.41 (m, 2H), 7.37-7.33 (m, 2H), 6.53 (s, 1H),4.98 (t, J=5.0, 1H), 4.55-4.54 (m, 2H), 4.45-4.44 (m, 2H), 4.13-4.32 (m,5H), 3.91 (s, 1H), 3.43-3.41 (m, 1H), 3.10 (s, 4H), 2.60-2.38 (m, 8H),1.78-1.76 (m, 2H), 1.69-1.67 (m, 2H)

Example 109a 2-Bromo-4-chloro-6-nitrobenzenamine 109a

A mixture of 4-chloro-2-nitrobenzenamine (5.1 g, 30 mmol) andN-bromosuccinimide (6.2 g, 36 mmol) in acetonitrile (50 mL) was heatedat reflux for overnight. It was cooled to room temperature and dilutedwith ethyl acetate (50 mL). The mixture was washed with saturatedaqueous K₂CO₃ solution (100 mL×2). The organic phase was separated,dried over anhydrous Na₂SO₄, filtered, and evaporated under reducedpressure to afford 109a as a yellow solid (8.1 g, 100%). LCMS: [M+H]⁺253. ¹H NMR (500 MHz, CDCl₃) δ 8.17 (d, J=2.5 Hz, 1H), 7.71 (d, J=2.5Hz, 1H), 6.64 (s, 2H).

Example 109b 3-Bromo-5-chlorobenzene-1,2-diamine 109b

To a solution of 109a (5.0 g, 20 mmol) in ethyl acetate (100 mL) wasadded SnCl₂.2H₂O (22.6 g, 100 mmol). The reaction was heated at refluxfor 2 h. After cooled to room temperature, the reaction mixture waspoured into aqueous Na₂CO₃ (200 mL) and the mixture was extracted withethyl acetate (200 mL×3). The combined organic phase was dried overanhydrous Na₂SO₄, filtered, and evaporated under reduced pressure toafford 109b as a dark red liquid (4.3 g, 97%). LCMS: [M+H]⁺ 223. ¹H NMR(500 MHz, CDCl₃) δ 6.97 (d, J=2.5 Hz, 1H), 6.65 (d, J=2.5 Hz, 1H), 3.67(bs, 4H).

Example 109c 4-Bromo-6-chloro-1H-benzo[d]imidazole 109c

A mixture of 109b (4.3 g, 19.5 mmol) in formic acid (10 mL) was heatedat 80° C. for 1 h. After cooled to room temperature, the reactionmixture was poured into aqueous Na₂CO₃ (30 mL) and the mixture wasextracted with ethyl acetate (30 mL×3). The combined organic phase wasdried over anhydrous Na₂SO₄, filtered, and evaporated under reducedpressure to afford 109c as a brown solid (3.5 g, 77%). LCMS: [M+H]⁺ 233.¹H NMR (500 MHz, CDCl₃) δ 10.05 (bs, 1H), 8.18 (s, 1H), 7.64 (bs, 1H),7.50 (s, 1H).

Example 109d 4-Bromo-6-chloro-1-methyl-1H-benzo[d]imidazole 109d

To a solution of 109c (3.5 g, 15 mmol) in N,N-dimethylformamide (30 mL)was added potassium carbonate (4.14 g, 30 mmol) and iodomethane (1.4 mL,22.7 mmol). The reaction was stirred at room temperature for overnight.Water (50 mL) was added to quench the reaction and the resulting mixturewas extracted with ethyl acetate (5 mL×3). The combined organic phasewas dried over anhydrous Na₂SO₄, filtered, and evaporated under reducedpressure. The residue was purified by column chromatography eluting withpetroleum ether/ethyl acetate (from 4:1 to 1:2) to afford 109d as awhite solid (1.4 g, 38%). LCMS: [M+H]⁺ 245/247. ¹H NMR (500 MHz, CDCl₃)δ 7.89 (s, 1H), 7.46 (d, J=2.0 Hz, 1H), 7.32 (d, J=2.0 Hz, 1H), 3.81 (s,3H).

Example 109e6-Chloro-1-methyl-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)-1H-benzo[d]imidazol-4-amine109e

A microwave vial equipped with a magnetic stirrer was charged with 109d(486 mg, 2.0 mmol), 5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-amine(390 mg 1.67 mmol), cesium carbonate (1.09 g, 3.34 mmol),tris(dibenzylideneacetone)dipalladium(0) (152 mg, 0.167 mmol) andXantphos (193 mg, 0.334 mmol). After bubbling nitrogen through thesuspension for 5 minutes, the reaction was heated at 120° C. forovernight. It was then cooled to room temperature and filtered. Thefiltrate was concentrated under reduced pressure and the residue waswashed with a mixture of petroleum ether/ethyl acetate (15 mL, 2:1) toafford 109e as a yellow solid (720 mg, 100%). LCMS: [M+H]⁺ 399.3. ¹H NMR(500 MHz, CDCl₃) δ 8.20 (d, J=2.0, 1H), 8.04 (d, J=2.5, 1H), 7.72 (s,1H), 7.61 (s, 1H), 7.28-7.29 (m, 1H), 6.90-6.91 (m, 2H), 4.70-4.71 (m,4H), 3.77 (s, 3H), 3.70-3.71 (m, 1H), 3.17 (t, J=5.0, 4 H), 2.52 (t,J=5.0, 4H)

Example 1092-(5-Fluoro-2-(hydroxymethyl)-3-(3-methyl-7-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-3H-benzo[d]imidazol-5-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one109

A microwave vial equipped with a magnetic stirrer was charged with 109e(300 mg, 0.75 mmol),4-fluoro-2-(1-oxo-3,4,6,7,8,9-Hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylacetate 101l (545 mg, 1.13 mmol), potassium carbonate (414 mg, 3.0mmol), tris(dibenzylideneacetone)dipalladium(0) (68 mg, 0.075 mmol), andtricyclohexylphosphine (210 mg, 0.75 mmol). After bubbling nitrogenthrough the suspension for 5 minutes, the reaction was heated at 110° C.for overnight. It was then cooled to room temperature and filtered. Thefiltrate was concentrated under reduced pressure and the residue waspurified by reverse-phase prep-HPLC to afford 109 as a white solid (71mg, 14%). LCMS: [M+H]⁺ 677.3. ¹H NMR (500 MHz, DMSO) δ 8.64 (s, 1H),8.23 (d, J=1.0, 1H), 8.18 (s, 1H), 7.88 (d, J=2.5, 1H), 7.38-7.39 (m,1H), 7.33-7.35 (m, 1H), 7.24 (d, J=9.0, 1H), 7.18-7.19 (m, 2H), 6.52 (s,1H), 4.85 (bs, 1H), 4.56 (t, J=6.0, 2H), 4.46 (t, J=6.0, 2H), 4.29 (s,2H), 4.16-4.18 (m, 3H), 3.93-3.94 (m, 1H), 3.84 (s, 3H), 3.44-3.45 (m,1H), 3.07 (t, J=4.0, 4H), 2.61-2.62 (m, 2H), 2.47 (t, J=6.0, 2H), 2.39(t, J=4.0, 4H), 1.79-1.80 (m, 2H), 1.70-1.71 (m, 2H)

Example 110a (E)-Ethyl 3-(2-Chloro-4,4-dimethylcyclopent-1-enyl)acrylate110a

The following two procedures were adapted from Organic Preparations andProcedures Int., 29 (4), 471-498. A 500-mL single neck round bottomedflask equipped with a magnetic stirrer and nitrogen inlet was chargedwith 2-chloro-4,4-dimethylcyclopent-1-enecarbaldehyde (38 g, 240 mmol)in benzene (240 mL). To the solution was added ethoxycarbonylmethylenetriphenylphosphorane (84 g, 240 mmol). The mixture was stirred for 14 h.After that time, the solvent was evaporated and the residue wastriturated with hexanes (2 L) to extract the product away from the PPh₃by-products. The organic layer was dried over sodium sulfate andconcentrated in vacuo. The residue was purified by column chromatographyusing a 100% hexane-1:1 hexane/ethyl acetate gradient to afford a 37%yield (20 g) of 110a.

Example 110b Ethyl5,5-Dimethyl-1,4,5,6-tetrahydrocyclopenta[b]pyrrole-2-carboxylate 110b

A 250-mL single neck round bottomed flask equipped with a magneticstirrer and nitrogen inlet was charged with 110a (17 g, 74 mmol) in DMSO(100 mL). To the solution was added sodium azide (9.6 g, 150 mmol). Themixture was then heated to 75° C. and stirred for 8 h. After cooling tort, H₂O (100 mL) and CH₂Cl₂ (200 mL) were added and the organic layerwas separated. The aqueous layer was extracted with CH₂Cl₂ (50 mL). Thecombined organic layers were washed with brine, dried over sodiumsulfate and concentrated in vacuo. The residue was purified by columnchromatography using a 100% hexane-1:1 hexane/ethyl acetate gradient toafford a 37% yield (5.7 g) of 110b.

Example 110c Ethyl1-(Cyanomethyl)-5,5-dimethyl-1,4,5,6-tetrahydrocyclopenta[b]pyrrole-2-carboxylate110c

A 250-mL single neck round bottomed flask equipped with a magneticstirrer and nitrogen inlet was charged with 110b (6.2 g, 30 mmol) in DMF(57 mL). To the solution was added NaH (80% dispersion in mineral oil,1.26 g, 42.1 mmol). The resulting mixture was stirred at rt for 90 min.After that time, bromoacetonitrile (2.94 mL, 42 mmol) was added. Themixture was stirred for 14 h. After that time, water (100 mL) and ethylacetate (200 mL) were added and the organic layer was separated. Theaqueous layer was extracted with ethyl acetate (2×50 mL). The combinedorganic layers were washed with brine, dried over sodium sulfate andconcentrated in vacuo. The residue was purified by column chromatographyto afford a 95% yield (7 g) of 110c.

Example 110d Ethyl1-(2-Aminoethyl)-5,5-dimethyl-1,4,5,6-tetrahydrocyclopenta[b]pyrrole-2-carboxylatehydrochloride 110d

A 500-mL Parr reactor bottle was purged with nitrogen and charged with10% palladium on carbon (50% wet, 2.0 g dry weight), 110c (4.5 g, 18mmol), 12% hydrochloric acid (9.2 mL, 37 mmol), ethyl acetate (80 mL)and ethanol (52 mL). The bottle was attached to a Parr hydrogenator,evacuated, charged with hydrogen gas to a pressure of 50 psi and shakenfor 6 h. After this time, the hydrogen was evacuated, and nitrogen wascharged into the bottle. Celite 521 (10.0 g) was added, and the mixturewas filtered through a pad of Celite 521. The filter cake was washedwith ethanol (2×50 mL), and the combined filtrates were concentrated todryness under reduced pressure. The crude residue 110d was carried ontothe next step without further purification.

Example 110e4,4-Dimethyl-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-9-one110e

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and nitrogen inlet was purged with nitrogen and charged withcrude 110d (˜18 mmol), sodium ethoxide (6.2 g, 92 mmol) and ethanol (120mL). The mixture was stirred at 55° C. over night. After that time, thereaction mixture was concentrated under reduced pressure and the residuewas partitioned between ethyl acetate (200 mL) and water (100 mL). Thesolution was filtered. The solid was washed with ethyl acetate (15 mL)to give 850 mg of desired product 110e. The organic layer was separated,and the aqueous layer was extracted with ethyl acetate (2×100 mL). Thecombined organic layers were dried over sodium sulfate and concentratedunder reduced pressure to near dryness. The solution was filtered andthe solid (1.44 g) was washed with ethyl acetate (15 mL). The combinedsolids were dried under vacuum a afford 61% yield (2.3 g) of 110e.

Example 110f2-Bromo-4-fluoro-6-(9-oxo-4,4-dimethyl-1,10diazatricyclo[6.4.0.0^(2,6)]-dodeca-2(6),7-dien-10-yl)benzylAcetate 110f

A sealed tube was equipped with a magnetic stirrer and charged with 110e(740 mg, 3.6 mmol), 2,6-dibromo-4-fluorobenzyl acetate 101j (2.4 g, 7.2mmol) and cesium carbonate (2.6 g, 7.9 mmol) in 1,4-dioxane (36 mL).After bubbling nitrogen through the solution for 30 min, Xantphos (250mg, 0.43 mmol) and tris(dibenzylideneacetone) dipalladium(0) (260 mg,0.29 mmol) were added, and the reaction mixture was heated to 100° C.for 16 h. After this time, H₂O (50 mL) and ethyl acetate (50 mL) wereadded. The aqueous layer was separated and extracted with ethyl acetate(2×50 mL). The combined organic extracts were washed with brine (100 mL)and dried over sodium sulfate. The resulting residue was purified bycolumn chromatography eluting with a gradient of 100% hexanes-100% Ethylacetate to afford a 56% yield (910 mg) of 110f.

Example 110g2-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-4-fluoro-6-(9-oxo-4,4-dimethyl-1,10diazatricyclo[6.4.0.0^(2,6)]-dodeca-2(6),7-dien-10-yl)benzylAcetate 110g

Compound 110g was synthesized using the same procedure as 103g, exceptusing 110f (450 mg, 1.0 mmol),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (635 mg, 2.5mmol), potassium acetate (393 mg, 4.0 mmol),bis(diphenylphosphino)-ferrocene]dichloropalladium(II) complex withCH₂Cl₂ (Pd Cl₂dppf:CH₂Cl₂ (1:1), 66 mg, 0.08 mmol) and 1,4-dioxane (20mL). The reaction mixture was heated at 100° C. for 5 h. The reactionmixture was cooled to room temperature and filtered through a pad ofCelite 521. The filter cake was washed with Ethyl Acetate (2×25 mL), andthe combined filtrates were concentrated to dryness under reducedpressure to afford 110g (quantitative yield) as black oil, which wasused directly for the next step. MS (ESI+) m/z 497.3 (M+H).

Example 11010-[5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-9-one110

To a suspension of6-chloro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)imidazo[1,2-a]pyridin-8-amine102c (300 mg, 0.777 mol) in dioxane/H₂O (12 mL/1 mL) was added 110g (385mg, 0.777 mmol), Pd₂(dba)₃ (70 mg, 0.0777 mmol), tricyclohexylphosphine(220 mg, 0.777 mmol), and CsCO₃ (510 mg, 1.554 mmol). This mixture wasstirred at 120° C. under N₂ for 15 h. It was then filtered and thefiltrate was concentrated to give a yellow solid, which was furtherpurified by reverse-phase prep-HPLC to afford 110 as a white solid (70mg, 15% yield). LCMS: [M+H]⁺ 678. ¹H NMR (500 MHz, MeOD) δ 8.29 (s, 1H),8.09 (d, J=2.5, 1H), 8.05 (s, 1H), 7.66 (s, 1H), 7.50 (dd, J=3.5, 9.5,1H), 7.41 (dd, J=2.0, 9.0, 1H), 7.34 (dd, J=2.5, 8.5, 1H), 7.17 (d, J=9,1H), 6.70 (s, 1H), 4.74 (t, J=7, 2H), 4.65 (t, J=6, 2H), 4.58-4.59 (m,2H), 4.29-4.30 (m, 1H), 4.24-4.25 (m, 2H), 4.04-4.05 (m, 1H), 3.58-3.59(m, 1H), 3.26-3.27 (m, 4H), 2.61 (s, 2H), 2.56-2.58 (m, 4H), 2.50 (s,2H), 1.27 (s, 6H)

Example 1115-[5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)phenyl]-8-thia-5-azatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7)-dien-6-one111

A sealed tube equipped with a magnetic stirrer was charged with(4-fluoro-2-{6-oxo-8-thia-5-azatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methylacetate 103g (200 mg, 0.44 mmol),6-chloro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)imidazo[1,2-b]pyridazin-8-amine108a (155 mg, 0.44 mmol), tricyclohexylphosphine (112 mg, 0.4 mmol),Pd2(dba)₃ (28 mg, 0.024 mmol), Cs2CO3 (261 mg, 0.8 mmol), and dioxane(25 mL). After three cycles of vacuum/argon flash, the reaction mixturewas heated at 110° C. for 16 h. The mixture was then evaporated in vacuoand the residue was extracted with Ethyl acetate (10 mL×2). The combinedEthyl acetate extract was concentrated under reduced pressure and theresidue was purified with reverse-phase prep-HPLC to afford 111 (59 mg,22%). MS: [M+H]⁺ 681. ¹H NMR (500 MHz, DMSO) δ 9.94 (s, 1H), 8.23 (s,1H), 8.16 (s, 1H), 8.02 (s, 1H), 7.67 (s, 1H), 7.44-7.46 (m, 3H),7.34-7.36 (m, 1H), 4.64-4.66 (m, 1H), 4.54-4.57 (m, 2H), 4.42-4.48 (m,3H), 4.35-4.38 (m, 1H), 3.96-4.10 (m, 1H), 3.79-3.89 (m, 1H), 3.42-3.45(m, 1H), 3.14-3.16 (m, 4H), 2.54-2.96 (m, 4H), 2.47-2.50 (m, 2H),2.37-2.41 (m, 4H), 1.79 (t, J=4.0, 4H)

Example 1125-[5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)phenyl]-8-thia-4,5-diazatricyclo-[7.4.0.0^(2,7)]trideca-1(9),2(7),3-trien-6-one112

A sealed tube equipped with a magnetic stirrer was charged with(4-fluoro-2-{6-oxo-8-thia-4,5-diazatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7),3-trien-5-yl}-6-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methylacetate (200 mg, 0.44 mmol),6-chloro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)imidazo[1,2-b]pyridazin-8-amine105a (155 mg, 0.44 mmol), tricyclohexylphosphine (112 mg, 0.4 mmol),Pd2(dba)₃ (28 mg, 0.024 mmol), Cs2CO3 (261 mg, 0.8 mmol), and dioxane(25 mL). After three cycles of vacuum/argon flush, the reaction mixturewas heated at 110° C. for 16 h. The mixture was evaporated in vacuo andthe residue was extracted with ethyl acetate (10 mL×2). The combinedethyl acetate extract was concentrated under reduced pressure and theresidue was purified with reverse-phase prep-HPLC to afford 112 (26 mg,12%). MS: [M+H]⁺ 680. ¹H NMR (500 MHz, DMSO) δ 9.95 (s, 1H), 8.49 (s,1H), 8.24 (s, 1H), 8.17 (s, 1H), 8.04 (s, 1H), 7.67 (s, 1H), 7.45-7.53(m, 4H), 4.54-4.57 (m, 3H), 4.45-4.47 (m, 2H), 4.33 (d, J=12.5, 2H),3.35-3.46 (m, 1H), 3.13-3.17 (m, 4H), 2.83-2.94 (m, 4H), 2.47-2.50 (m,4H), 1.82-1.87 (m, 4H)

Example 113a 3,3-Dimethylcyclopentanone 113a

A 1-L three-neck round-bottomed flask equipped with a magnetic stirrer,addition funnel and nitrogen inlet was purged with nitrogen and chargedwith ether (200 mL) and copper (I) iodide (54.46 g, 0.286 mol). Themixture was cooled to 0° C., methyllithium (1.6 M in ether, 357.5 mL,0.572 mol) was added dropwise to the reaction mixture over 1.5 h andstirred at 0° C. for additional 2 h. After this time a solution of3-methylcyclopent-2-enone (25 g, 0.260 mol) in ether (150 mL) was addeddropwise over 1.5 h. The reaction mixture was then stirred at 0° C. for2 h and poured into sodium sulfate deca-hydrate (300 g). The resultingmixture was stirred for 30 min. After this time the mixture was filteredand washed with ether (1000 mL). The filtrate was concentrated anddistilled under reduced pressure to afford a 70% yield (20.5 g) of3,3-dimethylcyclopentanone 113a as a colorless liquid: by 50-55° C. (at10 mmHg); ¹H NMR (300 MHz, CDCl₃) δ 2.31 (t, 2H, J=7.8 Hz), 2.05 (s,2H), 1.79 (t, 2H, J=7.8 Hz); MS (ESI+) m/z 113.3 (M+H)

Example 113b Ethyl5,5-Dimethyl-5,6-dihydro-4H-cyclopenta[b]thiophene-2-carboxylate 113b

A 500-mL three-neck round-bottomed flask equipped with a magneticstirrer, reflux condenser, addition funnel and nitrogen inlet was purgedwith nitrogen and charged with DMF (9.49 g, 0.100 mol) and methylenechloride (100 mL). The reaction mixture was cooled to 0° C. andphosphorus oxychloride (14.1 g, 0.0920 mol) was added dropwise to thereaction over 30 min. Once this addition was complete, the reaction waswarmed to room temperature and stirred for 1 h. After this time asolution of 113a (11.2 g, 0.100 mol) in methylene chloride (100 mL) wasadded dropwise over 1 h. The reaction was then stirred at reflux for 18h. The reaction mixture was cooled to room temperature and poured into amixture of crushed ice (400 mL) and sodium acetate (100 g, 1.22 mol).The resulting mixture was stirred for 45 min. After this time theaqueous layer was separated and extracted with methylene chloride (2×500mL). The combined organic layers were then washed with water (2×200 mL),followed by brine (200 mL) and dried over sodium sulfate. The dryingagent was then removed by filtration, and the filtrate was concentratedto afford crude product 2-chloro-4,4-dimethylcyclopent-1-enecarbaldehydewhich was placed in a 500-mL three-neck round bottomed flask equippedwith a mechanical stirrer, reflux condenser and nitrogen inlet.Methylene chloride (200 mL), ethyl 2-mercaptoacetate (11.0 g, 0.092 mol)and triethylamine (30 g, 0.207 mol) were then added. The reactionmixture was then stirred at reflux for 6 h. After this time the reactionwas cooled to room temperature and concentrated to a thick orangeresidue. Ethanol (200 mL) and triethylamine (30.0 g, 0.207 mol) wereadded and the reaction was heated at reflux for 12 h. The reaction wasthen cooled to room temperature and concentrated under reduced pressureand the resulting residue was diluted with ether (600 mL). The resultingmixture was washed with 1 M hydrochloric acid (150 mL), brine (100 mL),dried over sodium sulfate, filtered and concentrated under reducedpressure. The resulting residue was purified by flash chromatography toafford 113b in 34% yield (7.70 g) as a colorless liquid: ¹H NMR (300MHz, CDCl₃) δ 7.48 (s, 1H), 4.33 (q, 2H, J=7.2 Hz), 2.72 (s, 2H), 2.56(s, 2H), 1.38 (t, 3H, J=1.8 Hz), 1.17 (s, 6H); MS (ESI+) m/z 225.1

Example 113c5,5-Dimethyl-5,6-dihydro-4H-cyclopenta[b]thiophene-2-carboxylic acid113c

In a 250-mL single-neck round-bottomed flask equipped with a magneticstirrer and reflux condenser, 113b (4.00 g, 17.8 mmol) was dissolved inethanol (50 mL). THF (50 mL), water (50 mL) and lithium hydroxide (854mg, 35.6 mmol) were added, and the mixture was stirred at 60° C. for 4h. After this time the reaction was cooled to room temperature andacidified with 2M hydrochloric acid to pH 1.5, and then extracted withethyl acetate (2×200 mL). The organic layers were combined, washed withwater (2×100 mL), followed by brine (100 ml) and dried over sodiumsulfate. The drying agent was then separated by filtration. Afterevaporating the resulting filtrate, 113c was obtained in 91% yield (3.2g) as a white solid: mp 170-172° C.; ¹H NMR (300 MHz, CDCl₃) δ 12.77 (s,1H), 7.46 (s, 1H), 2.71 (s, 2H), 2.53 (s, 2H), 1.20 (s, 6H); MS (ESI−)m/z 195.0

Example 113d5,5-Dimethyl-5,6-dihydro-4H-cyclopenta[b]thiophene-2-carboxylic acid113d

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer, reflux condenser and a bubbler placed on the condenser wascharged with 113c (2.30 g, 11.6 mmol), toluene (25 mL), thionyl chloride(4.09 g, 34.9 mmol) and DMF (1 drop). The mixture was heated at refluxfor 1 h and then evaporated under reduced pressure on a rotaryevaporator at 45° C. The resulting acid chloride was diluted withmethylene chloride (20 mL).

In a separate 250-mL three-neck round-bottomed flask equipped with amagnetic stirrer N,O-dimethylhydroxylamine hydrochloride (2.26 g, 23.2mmol) and N,N-diisopropylethylamine (2.97 g, 23.0 mmol) were dissolvedin anhydrous methylene chloride (20 mL) under nitrogen, and the solutionwas cooled to 0° C. in an ice/water bath. The solution of the acidchloride was added, and the reaction mixture was stirred at roomtemperature for 18 h. The reaction mixture was extracted with water (100mL), 10% aqueous citric acid (50 mL) and a 1:1 mixture of saturatedaqueous sodium bicarbonate and water (100 mL). The organic layer wasdried over sodium sulfate and evaporated under reduced pressure on arotary evaporator to afford a 93% yield (2.60 g) of 113d as a lightyellow solid: ¹H NMR (300 MHz, CDCl₃) δ 7.66 (s, 1H), 3.77 (s, 3H), 3.35(s, 3H), 2.74 (s, 2H), 2.58 (s, 2H), 1.23 (s, 6H)

Example 113e3-Chloro-1-(5,5-dimethyl-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)propan-1-one113e

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer was purged with nitrogen and charged with 113d (2.41 g, 10.0mmol) and anhydrous THF (20 mL). The solution was cooled to −70° C., and1 M vinylmagnesium bromide in THF (11 mL, 11.0 mmol) was added with thereaction temperature maintained below −60° C. The reaction mixture wasstirred at −13 to −7° C. for 2 h and then warmed to room temperatureover 30 min. The reaction was again cooled to −70° C., and a 2 Msolution of hydrogen chloride in ether (22.5 ml, 45 mmol) was added. Thereaction was then stored in a freezer at −10° C. overnight. After thistime the mixture was evaporated under reduced pressure on a rotaryevaporator, and the resulting residue partitioned between water (100 mL)and ether (100 mL). The ether extract was dried over sodium sulfate andevaporated under reduced pressure on a rotary evaporator to afford crude113e (2.86 g, 118%) as a brown oil with approximately 75% purity (byNMR): ¹H NMR (300 MHz, CDCl₃) δ 7.45 (s, 1H), 3.89 (t, 2H, J=6.9 Hz),3.30 (t, 2H, J=6.9 Hz), 2.75 (s, 2H), 2.59 (s, 2H), 1.24 (s, 6H)

Example 113f6,6-Dimethyl-1,2,6,7-tetrahydrodicyclopenta[b,d]thiophen-3(5H)-one 113f

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer was charged with crude 113e (2.86 g, 10.0 mmol presumingquantitative yield) and 98% sulfuric acid. The reaction mixture washeated in a 90° C. oil bath overnight. The reaction mixture was placedinto an ice/acetone bath, and a cold (5° C.) solution of dipotassiumhydrogen phosphate (105 g, 0.603 mol) in water (300 mL) was added in oneportion. The resulting mixture was shaken with ethyl acetate (300 mL)and filtered. The filter cake was washed with ethyl acetate (100 mL).The ethyl acetate layer of the filtrate was separated, dried over sodiumsulfate and evaporated under reduced pressure on a rotary evaporator.the resulting residue was purified by flash column chromatography(silica, 80:20 hexanes/ethyl acetate) to afford 113f in 37% yield overtwo steps (683 mg) as an amorphous brown solid: mp 60-62° C.; ¹H NMR(500 MHz, CDCl₃) δ 2.92-2.87 (m, 4H), 2.79 (s, 2H), 2.53 (s, 2H), 1.26(s, 6H); MS (ESI+) m/z 207.0 (M+H)

Example 113g6,6-Dimethyl-1,2,6,7-tetrahydrodicyclopenta[b,d]thiophen-3(5H)-one 113g

A 250-mL single-neck round-bottomed flask equipped with a magneticstirrer and nitrogen inlet was charged with hydroxylamine hydrochloride(688 mg, 9.90 mmol), sodium acetate (812 mg, 9.90 mmol) and methanol (10mL), and the mixture at room temperature for 30 min. After this time, asolution of 113f (680 mg, 3.30 mmol) was added dropwise at roomtemperature, and the reaction was stirred at room temperature for 14 hunder nitrogen atmosphere. Since the reaction was not complete,hydroxylamine hydrochloride (1.15 g, 16.5 mmol) and sodium acetate (1.35g, 16.5 mmol) were added, and the stirring was continued at roomtemperature for 58 h. After this time, the mixture was diluted withmethylene chloride (150 mL) and water (100 mL), and the layers wereseparated. The organic layer was washed with brine (50 mL) and driedover sodium sulfate. The drying agent was removed by filtration and thefiltrate was concentrated to afford crude 113g in quantitative yield(730 mg) as a yellow semi-solid which was used in the next step withoutpurification: mp 122-124° C.; ¹H NMR for major isomer (500 MHz, CDCl₃) δ3.13-3.11 (m, 2H), 2.85-2.83 (m, 2H), 2.77 (s, 2H), 2.49 (s, 2H), 1.24(s, 6H); MS (ESI+) m/z 222.0 (M+H)

Example 113h6,6-Dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridine-1(2H)-one113h

A 100-mL three-neck round-bottomed flask equipped with a refluxcondenser, mechanical stirrer and nitrogen inlet was charged with 113g(700 mg, 3.16 mmol) and polyphosphoric acid (25 g). The reaction mixturewas stirred at 80° C. for 13 h under nitrogen atmosphere. After thistime, the mixture was cooled to 0° C. and water (50 mL) was addeddropwise carefully maintaining the internal temperature between 10-45°C. The mixture was diluted with 90:10 methylene chloride/methanol (100mL) and the layers were separated. The aqueous layer was extracted with90:10 methylene chloride/methanol (50 mL), and the combined organiclayers were washed with saturated aqueous sodium bicarbonate (50 mL),brine (150 mL) and dried over sodium sulfate. The drying agent wasremoved by filtration. The filtrate was concentrated under reducedpressure, and the resulting residue was purified by flash columnchromatography (silica, 95:5 methylene chloride/methanol) to afford6,6-dimethyl-3,4,6,7-tetrahydro-5H-cyclopenta[4,5]thieno[2,3-c]pyridine-1(2H)-one113h in 90% yield (630 mg) as an amorphous off-white solid: mp 205-207°C.; ¹H NMR (500 MHz, CDCl₃) δ 5.51 (s, 1H), 3.60-3.56 (m, 2H), 2.76-2.73(m, 4H), 2.49 (s, 2H), 1.26 (s, 6H); MS (ESI+) m/z 222.0 (M+H)

Example 113i(2-Bromo-6-{4,4-dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0^(2,6)]dodeca-1(8),2(6)-dien-10-yl}-4-fluorophenyl)methylAcetate 113i

A mixture of 113h (2 g, 9.05 mmol), 2,6-dibromo-4-fluorobenzyl acetate(101j)(8.8 g, 27.15 mmol), XantPhos (524 mg, 0.9 mmol), Pd₂(dba)₃ (828mg, 0.9 mmol) and Cs₂CO₃ (5.9 g, 18 mmol) in dioxane (200 mL) was heatedat 100° C. for 15 h under nitrogen. The reaction mixture was filteredand the filtrated was evaporated in vacuo. The residue was purified bysilical-gel column eluting with 1:1 ethyl acetate/petroleum ether togive 113i as a yellow solid (3 g, 71%). MS: (M+H)⁺ 466.

Example 113j(2-{4,4-Dimethyl-9-oxo-7-thia-10-azatricyclo[6.4.0.0^(2,6)]dodeca-1(8),2(6)-dien-10-yl}-4-fluoro-6-(tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methylAcetate 113j

A solution of 113i (3 g, 6.45 mmol),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane) (9.8 g, 38.7mmol) in dioxane (160 mL) was added PdCl₂(dppf) (525 mg, 0.65 mmol) andCH₃COOK (3.8 g, 38.7 mmol). The mixture was stirred at 100° C. for 15 hunder argon atmosphere. After reaction the mixture was filtered andevaporated in vacuo and the residue was purified by silical-gel columneluting with 1:2 ethyl acetate/petroleum ether to give 113j as a yellowsolid (2.5 g, 76%). MS: (M+H)⁺ 514.

Example 11310-[5-Fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridine-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0^(2,6)]dodeca-1(8),2(6)-dien-9-one113

Following the procedures in Example 112,6-chloro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)imidazo[1,2-a]pyridin-8-amineand 113j were reacted to give 113 in 13% yield. LCMS: [M+H]⁺ 695. ¹H NMR(500 MHz, MeOD) δ 8.30 (s, 1H), 8.09 (d, J=2.5, 1H), 8.05 (s, 1H), 7.66(s, 1H), 7.50 (dd, J=3.5, 9.0, 1H), 7.40 (dd, J=3, 9, 1H), 7.34 (dd,J=2, 9, 1H), 7.16 (d, J=9, 1H), 4.74 (t, J=6.5, 2H), 4.65 (t, J=6, 2H),4.60-4.61 (m, 2H), 4.16-4.17 (m, 1H), 4.01-4.02 (m, 1H), 3.58-3.59 (m,1H), 3.25-3.26 (m, 4H), 3.12-3.14 (m, 1H), 2.97-2.98 (m, 1H), 2.81 (s,2H), 2.60-2.61 (m, 2H), 2.55-2.56 (m, 4H), 1.29 (d, J=2.5, 6H)

Example 114a 2-Bromo-4-chloronicotinaldehyde 114a

To a solution of 2-bromo-4-chloropyridine (1.6 g, 8.0 mmol) in anhydroustetrahydrofuran (40 mL) cooled at −70° C. was added the solution oflithium diisopropyl-amide (5.0 mL, 10.0 mmol, 2.0 M) over a period of 5minutes and stirred at −70° C. for another 1 h. Anhydrous DMF (1.3 g)was introduced over a period of 3 minutes and the mixture was stirredfor another 30 minutes. It was then quenched with saturated NH₄Cl (30mL) and extracted with ethyl acetate (20 mL×3). The combined organiclayer was dried over anhydrous Mg₂SO₄, filtered, and evaporated underreduced pressure. The residue was purified by silica-gel columnchromatography eluting with petroleum ether/ethyl acetate (20:1) toafford 114a as a yellow solid (900 mg, 48%). ¹H NMR (500 MHz, DMSO) δ10.21 (s, 1H), 8.52 (d, J=5.5 Hz, 1H), 7.79 (d, J=5.0 Hz, 1H).

Example 114b4-Chloro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)nicotinaldehyde114b

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 114a (800 mg, 3.5 mmol),3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one 101g (665 mg, 3.5mmol), tris(dibenzylideneacetone)dipalladium(0) (320 mg, 0.35 mmol),XantPhos (400 mg, 0.7 mmol), Cs₂CO₃ (2.3 g, 7.0 mmol), and 1,4-dioxane(20 mL). After three cycles of vacuum/argon flush, the mixture washeated at 90° C. for 5 h. It was then cooled to room temperature andfiltered. The filtrate was concentrated under reduced pressure and theresulting residue was purified by silica-gel column chromatographyeluting with dichloromethane/methanol (80:1) to afford 114b as yellowsolid (1.2 g, 50%). MS: [M+H]⁺ 330.

Example 114c2-(4-chloro-3-(hydroxymethyl)pyridin-2-yl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one114c

To a solution of 114b (1.0 g, 3.0 mmol) in methanol (50 mL) was addedsodium borohydride (380 mg, 9.0 mmol) at 10° C. and stirred for another30 minutes. Then the reaction mixture was quenched with water (1 mL) andconcentrated. The residue was dissolved in dichloromethane (50 mL) andwashed with water (10 mL). The organic phase was dried over anhydrousNa₂SO₄, filtered, and evaporated under reduced pressure to afford 114cas a yellow solid (900 mg, 90%). MS: [M+H]⁺ 332.

Example 114d(4-Chloro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)pyridine-3-yl)methylacetate 114d

To a mixture of 114c (900 mg, 2.7 mol) and triethylamine (900 mg, 9.0mol) in dichloromethane (5 mL) was added dropwise acetyl chloride (600mg, 6.0 mol) while stirring at room temperature and stirred for another1 h. The reaction mixture was concentrated and purified by silica-gelcolumn chromatography eluting with dichloromethane to afford 114d aswhite solid (950 mg, 94%). MS: [M+H]⁺ 374.

Example 114e(2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-3-yl)methylacetate 114e

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 114d (950 mg, 2.5 mmol),Pd₂B₂ (1.6 g, 2.0 eq., 5 mmol), Pd₂(dba)₃ (230 mg, 0.1 eq., 0.25 mmol),X-phos (232 mg, 0.2 eq., 0.5 mmol), AcOK (735 mg, 3 eq., 7.5 mmol) anddioxane (20 mL). After three cycles of vacuum/argon flush, the mixturewas heated to 65° C. for 14 h. It was then cooled to room temperatureand filtered. The filtrate was concentrated under reduced pressure andthe resulting residue was washed by PE/EA=3/1 (10 mL) to afford 114e asyellow solid (950 mg, 87%). MS: [M+H]⁺ 383.

Example 1142-(3-(Hydroxymethyl)-4-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)pyridin-2-yl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one114

A sealed tube was charged with6-chloro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridine-2-yl)imidazo[1,2-b]pyridazin-8-amine105a (200 mg, 0.5 mmol), 114e (200 mg, 1.0 eq., 0.5 mmol), Pd₂(dba)₃ (46mg, 0.1 eq., 0.05 mmol), PCy₃ (40 mg, 0.2 eq., 0.1 mmol), cesiumcarbonate (325 mg, 2 eq., 1.0 mmol), and dioxane (10 mL). After threecycles of vacuum/argon flash, the mixture was heated at 130° C. for 14h. It was then cooled to room temperature and filtered. The filtrate wasconcentrated under reduced pressure. The resulting residue was purifiedby silica-gel column chromatography eluting withdichloromethane/methanol (40:1) and further purified with reverse-phaseprep-HPLC to afford 114 (13 mg, 4%). LCMS: [M+H]⁺ 647. ¹H NMR (500 MHz,DMSO) δ 9.96 (s, 1H), 8.56 (d, J=5.0, 1H), 8.24 (s, 1H), 8.18 (s, 1H),7.99 (d, J=2.5, 1H), 7.68 (s, 1H), 7.45-7.50 (m, 3H), 6.58 (s, 1H), 4.72(t, J=5.0, 1H), 4.55-4.59 (m, 3H), 4.45-4.47 (m, 2H), 4.37-4.40 (m, 1H),4.19-4.29 (m, 2H), 4.08-4.10 (m, 1H), 3.91-3.94 (m, 1H), 3.42-3.45 (m,1H), 3.14-3.16 (m, 4H), 2.52-2.63 (m, 2H), 2.46-2.47 (m, 2H), 2.36-2.40(m, 4H), 1.76-1.78 (m, 2H), 1.66-1.70 (m, 2H)

Example 115a tert-Butyl 4-(6-Nitropyridin-3-yl)piperazine-1-carboxylate115a

To a solution of 5-bromo-2-nitropyridine (30 g, 148 mmol) in DMSO (1 L)was added K₂CO₃ (40 g, 296 mmol) and tert-butyl piperazine-1-carboxylate(28 g, 148 mmol). The mixture was stirred at 65° C. overnight. Aftercooling down, it was poured into water (2 L). The precipitated solid wascollected and dried under vacuum. It was then further purified by flashcolumn eluting with 20:1 petroleum ether/ethyl acetate and then withmethylene chloride to give 115a as a yellow solid (17 g, 37%). MS:[M+H]⁺ 309.

Example 115b tert-Butyl 4-(6-Aminopyridin-3-yl)piperazine-1-carboxylate115b

A 500-mL bottle was purged with nitrogen and charged with 115a (3.1 g,10 mmol), 10% palladium on carbon (50% wet, 1.0 g) and ethanol (100 mL).It was evacuated, charged with hydrogen gas, and stirred for 16 h atroom temperature. The hydrogen was then evacuated and nitrogen wascharged into the bottle. The catalyst was removed by filtration througha pad of Celite and the filtrate concentrated under reduced pressure toafford 115b (2.7 g, 97%). MS: [M+H]⁺ 279

Example 115c (S)-tert-Butyl4-(6-(6-Chloroimidazo[1,2-b]pyridazin-8-ylamino)pyridin-3-yl)-3-methylpiperazine-1-carboxylate115c

Following the procedure for 101b and starting with8-bromo-6-chloroimidazo[1,2-b]pyridazine 104a (1.44 g, 6.2 mmol), and115b (905 mg, 3.1 mmol) afforded 115c as a yellow solid (2.2 g, 80%).MS-ESI: [M+H]⁺ 444.2

Example 115d(S)-6-Bromo-N-(5-(2-methylpiperazin-1-yl)pyridin-2-yl)imidazo[1,2-b]pyridazin-8-aminehydrobromide 115d

An autoclave was charged with 115c (1.60 g, 3.6 mmol) and HBr/AcOH (60mL). It was heated at 150° C. for 18 h. The reaction mixture wasconcentrated under reduced pressure to give black oil. The oil wasrecrystallized with methanol (30 mL)/dichloromethane (30 mL)/petroleumether (90 mL) to afford 115d as a yellow solid (1.1 g, 65%). MS-ESI:[M+H]⁺ 388.1

Example 115e(S)-6-Bromo-N-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)imidazo[1,2-b]pyridazin-8-amine115e

To a mixture of(S)-6-bromo-N-(5-(2-methylpiperazin-1-yl)pyridin-2-yl)imidazo[1,2-b]pyridazin-8-aminehydrobromide 115d (1.05 g, 2.24 mmol), oxetan-3-one (483 mg, 6.7 mmol),and ZnCl₂ (914 mg, 6.7 mmol) in methanol (30 mL) was added NaBH₃CN (421mg, 6.7 mmol). The reaction mixture was heated at 50° C. for 14 h andconcentrated under reduced pressure. Water (30 mL) was added to theresidue and the resulting mixture was extracted with dichloromethane (%X 30 mL). The combined organic phase was dried over anhydrous MgSO₄ andfiltered. The filtrate was evaporated under reduced pressure. Theresidue was purified by silica-gel column chromatography eluting with40:1 dichloromethane/methanol to afford 115e as a yellow solid (220 mg,22%). MS-ESI: [M+H]⁺ 444.1

Example 115(S)-2-(3-(hydroxymethyl)-4-(8-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)pyridin-2-yl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one115

A sealed tube was charged with 115e (156 mg, 0.35 mmol),3-(acetoxymethyl)-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)pyridin-4-ylboronicacid 114e (134 mg, 0.35 mmol), Pd₂(dba)₃ (64 mg, 0.070 mmol), PCy₃ (40mg, 0.14 mmol), cesium carbonate (228 mg, 0.70 mmol), water (1 drop),and dioxane (9 mL). After three cycles of vacuum/argon flush, themixture was heated at 130° C. for 16 h. It was then cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure and the resulting residue was purified by silica-gel columnchromatography eluting with 40:1 dichloromethane/methanol and furtherpurified with reverse-phase prep-HPLC to afford 115 (31 mg, 13%).MS-ESI: [M+H]⁺ 661.3. ¹H NMR (500 MHz, DMSO-d₆) δ 9.97 (s, 1H), 8.56 (d,J=5.0 Hz, 1H), 8.25 (d, J=5.0 Hz, 1H), 8.19 (d, J=0.5 Hz, 1H), 7.95 (s,1H), 7.68 (d, J=1.0 Hz, 1H), 7.48-7.44 (m, 3H), 6.58 (s, 1H), 4.76 (t,J=5.0 Hz, 1H), 4.58-4.54 (m, 3H), 4.49-4.46 (m, 1H), 4.43-4.36 (m, 2H),4.30-4.05 (m, 3H), 3.94-3.87 (m, 2H), 3.42-3.37 (m, 1H), 3.24-3.19 (m,1H), 3.00-2.96 (m, 1H), 2.66-2.57 (m, 3H), 2.47-2.43 (m, 3H), 2.28-2.26(m, 1H), 2.12-2.09 (m, 1H), 1.79-1.68 (m, 4H), 1.00 (d, J=6.5 Hz, 3H).

Example 116a4-Bromo-6-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[d]imidazole116a

To a solution of 4-bromo-6-chloro-1H-benzo[d]imidazole 109c (3.5 g, 15mmol) in N,N-dimethylformamide (30 mL) was added sodium hydride (360 mg,15 mmol) and 2-(trimethylsilyl)ethoxymethyl chloride (2.7 g, 16.5 mmol).The reaction was stirred at room temperature for 2 h. Water (100 mL) wasadded to quench the reaction. The mixture was extracted with ethylacetate (3×80 mL). The combined organic phase was dried over anhydrousNa₂SO₄, filtered, and evaporated under reduced pressure to afford 116aas a brown solid (4.2 g, 77%). MS-ESI: [M+H]⁺ 361.0

Example 116b6-Chloro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[d]imidazol-4-amine116b

A microwave vial equipped with a magnetic stirrer was charged with 116a(600 mg, 1.65 mmol), 5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-amine(390 mg 1.67 mmol), cesium carbonate (1.09 g, 3.34 mmol),tris(dibenzylideneacetone)dipalladium(0) (152 mg, 0.167 mmol), Xantphos(193 mg, 0.334 mmol), and dioxane (10 mL). After bubbling nitrogenthrough the suspension for 10 minutes, the reaction was heated at 120°C. overnight. It was then cooled to room temperature and filtered. Thefiltrate was concentrated under reduced pressure and the residue waswashed with a mixed solvent of petroleum ether and ethyl acetate (15 mL,2:1) to afford 116b as a yellow solid (720 mg, 85%). MS-ESI: [M+H]⁺515.2

Example 116c6-Chloro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)-1H-benzo[d]imidazol-4-amine116c

A mixture of 116b (720 mg, 1.4 mmol) in TFA (10 mL) was stirred at roomtemperature for 6 h. The mixture was concentrated under reduced pressureand the diluted with water (10 mL). The pH of the mixture was adjustedto 7 by adding aqueous ammonia. It was then extracted withdichloromethane (3×20 mL). The combined organic phase was dried overanhydrous Na₂SO₄, filtered, and evaporated under reduced pressure toafford 116c as a brown solid (500 mg, 93%). MS-ESI: [M+H]⁺ 385.1

Example 1162-[5-fluoro-2-(hydroxymethyl)-3-[7-[[5-[4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-3H-benzimidazol-5-yl]phenyl]-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1-one116

A microwave vial equipped with a magnetic stirrer was charged with 116c(300 mg, 0.78 mmol),4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylacetate 101k (545 mg, 1.13 mmol), potassium carbonate (414 mg, 3.0mmol), tris(dibenzylideneacetone)dipalladium(0) (68 mg, 0.075 mmol),tricyclohexyl phosphine (210 mg, 0.75 mmol), water (0.2 mL), and dioxane(10 mL). After bubbling nitrogen through the suspension for 10 minutes,the sealed vial was heated at 110° C. overnight. It was then cooled toroom temperature and filtered. The filtrate was concentrated underreduced pressure and the residue was purified by reverse-phase prep-HPLCto afford 116 as a white solid (52 mg, 10%). MS-ESI: [M+H]⁺ 663.3. ¹HNMR (500 MHz, DMSO) δ 8.23 (bs, 1H), 8.11 (s, 1H), 7.88 (d, J=4.5 Hz,1H), 7.35-7.31 (m, 1H), 7.23-7.17 (m, 2H), 7.13-7.09 (m, 2H), 6.52 (s,1H), 4.57-4.45 (m, overlap, 4H), 4.31 (d, J=7.5 Hz, 2H), 4.11-4.01 (m,4H), 3.51-3.47 (m, 1H), 3.10-3.06 (m, 4H), 2.59-2.41 (m, 8H), 1.81-1.68(m, 4H)

Example 117a6-Chloro-9-((2-(trimethylsilyl)ethoxy)methyl)-9H-purin-2-amine 117a

A mixture of 6-chloro-9H-purin-2-amine (5.0 g, 29.6 mmol) and NaH (1.43g, 32.5 mmol) in DMF (30 mL) was stirred at room temperature for 0.5 h.(2-(Chloromethoxy)ethyl)trimethylsilane (SEMCl, CAS Reg. No. 76513-69-4,4.91 g, 29.6 mmol) was added and the resulting mixture was stirred atroom temperature for 1.0 h. It was then filtered and the filtrate wasevaporated in vacuo. The residue was purified by silica-gel columnchromatography eluting with 3:1 petroleum ether/ethyl acetate to afford117a (5.1 g, 58%) as a yellow solid. MS-ESI: [M+H]⁺ 300.1

Example 117b6-Chloro-2-iodo-9-((2-(trimethylsilyl)ethoxy)methyl)-9H-purine 117b

To a mixture of 117a (2.99 g, 10.0 mmol), CH₂I₂ (4.0 ml, 51.0 mmol), andCuI (1.91 g, 10.0 mmol) in THF was added isoamyl nitrite (4.0 mL, 30.0mmol). The mixture was heated at reflux for 1.0 h and cooled to roomtemperature. The reaction mixture was partitioned between ethyl acetateand 1N aqueous HCl solution. The organic phase was washed with saturatedaqueous NH₄Cl, dried over Na₂SO₄, and concentrated under reducedpressure. The residue was purified by silica-gel column chromatographyeluting with 3:1 petroleum ether/ethyl acetate to afford 117b (2.02 g,49%) as a yellow solid. MS-ESI: [M+H]⁺ 411.0

Example 117c 1-(4-Nitrophenyl)piperazine 117c

To a solution of 117b (3.07 g, 10.0 mmol) in dioxane (50 mL) was added4.0M HCl/dioxane (10 mL, 40.0 mmol). The reaction mixture was stirred atroom temperature for 5 h. The mixture was then concentrated underreduced pressure to afford 117c (2.4 g, 99%) as a yellow solid, whichwas used without further purification. MS: [M+H]⁺ 208.

Example 117d 1-(4-Nitrophenyl)-4-(oxetan-3-yl)piperazine 117d

To a mixture of 117c (2.0 g, 8.23 mmol), zinc chloride (2.2 g, 16.4mmol), oxetan-3-one (1.18 g, 16.4 mmol) in methanol (80 mL) was addedNaBH₃CN (1.02 g, 16.4 mmol). The mixture was stirred at 50° C. for 5hours. The mixture was then concentrated under reduced pressure and theresidue was diluted with water (100 mL). It was extracted withdichloromethane (3×100 mL) and the combined organic layer wasconcentrated under reduced pressure. The residue was purified bysilica-gel column chromatography eluting with 4:1 petroleum ether/ethylacetate to afford 117d (1.65 g, 76%) as a yellow solid. MS: [M+H]⁺ 264.

Example 117e 4-(4-(Oxetan-3-yl)piperazin-1-yl)aniline 117e

A 250-mL round-bottomed flask was purged with nitrogen and charged with117d (1.5 g, 5.7 mmol), 10% palladium on carbon (50% wet, 750 mg), andethanol (60 mL). The flask was evacuated, charged with hydrogen gas, andstirred at room temperature for 15 h. The hydrogen was then evacuatedand nitrogen charged into the flask. The catalyst was removed byfiltration through a pad of Celite and the filtrate was concentratedunder reduced pressure to afford 117e (1.2 g, 90%), which was used inthe next step without further purification. MS: [M+H]⁺ 234

Example 117f2-Iodo-N-(4-(4-(oxetan-3-yl)piperazin-1-yl)phenyl)-9-((2-(trimethylsilyl)ethoxy)methyl)-9H-purin-6-amine117f

A mixture of 117b (2.05 g, 5.0 mmol), 117e (1.17 g, 5.0 mmol), andtriethylamine (1.01 g, 10 mmol) in i-propanol (30 mL) was stirred at 80°C. for 4 h. It was then filtered and the filtrate was evaporated invacuo. The residue was purified by silica-gel column chromatographyeluting with 30:1 dichloromethane/methanol to afford 117f (1.82 g, 60%)as a yellow solid. MS-ESI: [M+H]⁺ 608.1

Example 117g4-Fluoro-2-(6-(4-(4-(oxetan-3-yl)piperazin-1-yl)phenylamino)-9-((2-(trimethylsilyl)ethoxy)methyl)-9H-purin-2-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzylAcetate 117g

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 117f (607 mg, 1.0 mmol),4-fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylacetate 101k (482 mg, 1.0 mmol), Pd(dppf)Cl₂ (82 mg, 0.10 mmol), K₃PO₄(424 mg, 2.0 mmol), sodium acetate (164 mg, 2.0 mmol), water (0.5 mL),and acetonitrile (20 mL). After three cycles of vacuum/argon flush, themixture was heated at reflux for 2 h. It was then cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure and the resulting residue was purified by silica-gel columnchromatography eluting with 30:1 dichloromethane/methanol to afford 117gas yellow solid (600 mg, 72%). MS-ESI: [M+H]⁺ 836.5

Example 117h4-Fluoro-2-(6-(4-(4-(oxetan-3-yl)piperazin-1-yl)phenylamino)-9H-purin-2-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)benzylAcetate 117h

A mixture of 117g (550 mg, 0.660 mmol) and CF₃COOH (6 mL) was stirred atroom temperature for 2 h. It was then concentrated under reducedpressure to afford crude 117h as a yellow solid (140 mg, 30%), which wasused in the next step without further purification. MS-ESI: [M+H]⁺ 706.4

Example 1172-[5-fluoro-2-(hydroxymethyl)-3-[6-[4-[4-(oxetan-3-yl)piperazin-1-yl]anilino]-9H-purin-2-yl]phenyl]-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1-one117

A mixture of 117h (140 mg, 0.20 mmol) and lithium hydroxide (48 mg, 2.0mmol) in i-propanol/THF (1:1, 4 mL) and water (1 mL) was stirred at 30°C. for 1 h. The mixture was evaporated in vacuo and the residue wasdiluted with water (5 mL). It was then extracted with ethyl acetate(2×10 mL). The combined ethyl acetate extract was concentrated underreduced pressure and the residue was purified by reverse-phase prep-HPLCto afford 117 (85 mg, 64%) as a white solid. MS-ESI: [M+H]⁺ 664.4. ¹HNMR (500 MHz, CDCl₃) δ 12.45 (s, 1H), 8.26 (s, 1H), 7.86 (d, J=8.5 Hz,2H), 7.66 (d, J=8.0 Hz, 2H), 7.11 (d, J=9.0 Hz, 1H), 6.96 (d, J=8.0 Hz,2H), 6.88 (s, 1H), 4.74-4.68 (m, 4H), 4.66-4.60 (m, 2H), 4.17-4.09 (m,3H), 3.91-3.90 (m, 1H), 3.59 (t, J=6.5 Hz, 1H), 3.25-3.23 (m, 4H),2.59-2.52 (m, 8H), 1.89-1.80 (m, 4H).

Example 118a6-Chloro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[d]imidazol-4-amine118a

A microwave vial equipped with a magnetic stirrer was charged with4-bromo-6-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[d]imidazole116a (600 mg, 1.65 mmol),5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-amine (390 mg 1.67 mmol),cesium carbonate (1.09 g, 3.34 mmol),tris(dibenzylideneacetone)dipalladium(0) (152 mg, 0.167 mmol), Xantphos(193 mg, 0.334 mmol), and dioxane (10 mL). After bubbling nitrogenthrough the suspension for 10 minutes, the reaction was heated at 120°C. overnight. It was then cooled to room temperature and filtered. Thefiltrate was concentrated under reduced pressure and the residue waswashed with a mixed solvent of petroleum ether and ethyl acetate (15 mL,2:1) to afford 118a as a yellow solid (720 mg, 85%). MS-ESI: [M+H]⁺515.2

Example 118b6-Chloro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)-1H-benzo[d]imidazol-4-amine118b

A mixture of 118a (720 mg, 1.4 mmol) in TFA (10 mL) was stirred at roomtemperature for 6 h. The mixture was concentrated under reduced pressureand the diluted with water (10 mL). The pH of the mixture was adjustedto 7 by adding aqueous ammonia. It was then extracted withdichloromethane (3×20 mL). The combined organic phase was dried overanhydrous Na₂SO₄, filtered, and evaporated under reduced pressure toafford 118b as a brown solid (500 mg, 93%). MS-ESI: [M+H]⁺ 385.1

Example 1182-[3-(hydroxymethyl)-4-[7-[[5-[4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-3H-benzimidazol-5-yl]-2-pyridyl]-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1-one118

A microwave vial equipped with a magnetic stirrer was charged with 118b(200 mg, 0.52 mmol),3-(acetoxymethyl)-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)pyridin-4-ylboronicacid 114e (545 mg, 0.78 mmol), potassium carbonate (216 mg, 1.56 mmol),tris(dibenzylideneacetone) dipalladium(0) (48 mg, 0.052 mmol),tricyclohexylphosphine (146 mg, 0.52 mmol), water (0.2 mL), and dioxane(10 mL). After bubbling nitrogen through the suspension for 10 minutes,the sealed vial was irradiated under microwave at 110° C. for 1 h. Itwas then cooled to room temperature and filtered. The filtrate wasconcentrated under reduced pressure and the residue was purified byreverse-phase prep-HPLC to afford 118 as a white solid (50 mg, 15%).MS-ESI: [M+H]⁺ 646.3. ¹H NMR (500 MHz, DMSO) δ 8.69 (d, J=2.0 Hz, 1H),8.49 (d, J=5.0 Hz, 1H), 8.32-8.28 (m, 2H), 8.13 (s, 1H), 7.85-7.83 (m,1H), 7.39-7.33 (m, 2H), 7.36 (s, 1H), 7.29 (s, 1H), 6.59 (s, 1H), 5.89(s, 1H), 4.56 (t, J=6.5 Hz, 2H), 4.46 (t, J=6.5 Hz, 2H), 4.23-4.16 (m,2H), 4.15-4.12 (m, 4H), 3.89-3.53 (m, 1H), 3.13-3.07 (m, 5H), 2.64-2.54(m, 2H), 2.51-2.43 (m, 5H), 1.79-1.70 (m, 4H)

Example 119a{4-Fluoro-2-[6-({4-[4-(oxetan-3-yl)piperazin-1-yl]phenyl}amino)-9-{[2-(trimethylsilyl)ethoxy]methyl}purin-2-yl]-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7)-dien-5-yl}phenyl}methylAcetate 119a

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with(4-fluoro-2-{6-oxo-8-thia-5-azatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7)-dien-5-yl}-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)methylacetate 103g (288 mg, 0.60 mmol),2-iodo-N-(4-(4-(oxetan-3-yl)piperazin-1-yl)phenyl)-9-((2-(trimethylsilyl)ethoxy)methyl)-9H-purin-6-amine117f (364 mg, 0.60 mmol), Pd(dppf)Cl₂ (49 mg, 0.060 mmol), K₃PO₄ (254mg, 1.20 mmol), sodium acetate (98 mg, 1.20 mmol), water (0.5 mL), andacetonitrile (10 mL). After three cycles of vacuum/argon flush, themixture was heated at 100° C. for 2 h. It was then cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure and the resulting residue was purified by silica-gel columnchromatography eluting with 30:1 dichloromethane/methanol to afford 119a(307 mg, 60%). MS-ESI: [M+H]⁺ 853.4

Example 119b{4-Fluoro-2-[6-({4-[4-(oxetan-3-yl)piperazin-1-yl]phenyl}amino)-9H-purin-2-yl]-6-{6-oxo-8-thia-5-azatricyclo[7.4.0.0^(2,7)]trideca-1(9),2(7)-dien-5-yl}phenyl}methylAcetate 119b

A mixture of 119a (307 mg, 0.360 mmol) and CF₃COOH (5 mL) was stirred at30° C. for 2 h. The mixture was evaporated in vacuo and the residue wasdiluted with water (5 mL). The pH of the mixture was adjusted to 7 withsaturated NaHCO₃ solution and extracted with ethyl acetate (3×10 mL).The combined extract was dried over MgSO₄ and evaporated to dryness toafford 119b (150 mg, 60%), which was used in the next step withoutfurther purification. MS-ESI: [M+H]⁺ 723.4

Example 1192-[5-fluoro-2-(hydroxymethyl)-3-[6-[4-[4-(oxetan-3-yl)piperazin-1-yl]anilino]-9H-purin-2-yl]phenyl]-3,4,5,6,7,8-hexahydrobenzothiopheno[2,3-c]pyridin-1-one119

A mixture of 119b (150 mg, 0.210 mmol) and lithium hydroxide (50 mg,2.10 mmol) in i-propanol/THF (1:1, 4 mL) and water (1 mL) was stirred at30° C. for 1 h. The mixture was evaporated in vacuo and the residue wasdiluted with water (5 mL). It was then extracted with ethyl acetate(2×10 mL). The combined ethyl acetate extract was concentrated underreduced pressure and the residue was purified by reverse-phase prep-HPLCto afford 119 (85 mg, 58%) as a yellow solid. MS-ESI: [M+H]⁺ 681.3. ¹HNMR (500 MHz, CDCl₃) δ 12.72 (s, 1H), 8.45 (s, 1H), 7.93 (d, J=7.5 Hz,1H), 7.79 (s, 1H), 7.72 (d, J=9.0 Hz, 2H), 7.12 (d, J=8.0 Hz, 1H), 6.98(d, J=9.0 Hz, 2H), 4.74-4.67 (m, 6H), 4.09-4.04 (m, 1H), 3.78-3.73 (m,1H), 3.57 (t, J=6.5 Hz, 1H), 3.24-3.22 (m, 4H), 2.99-2.93 (m, 1H),2.88-2.83 (m, 3H), 2.53-2.51 (m, 6H), 1.93-1.81 (m, 5H).

Example 120a Methyl 5,6,7,8-Tetrahydroindolizine-2-carboxylate 120a

A 500-mL round-bottomed flask equipped with a magnetic stirrer andnitrogen inlet was purged with nitrogen and charged with5,6,7,8-tetrahydroindolizine-2-carboxylic acid (30.4 g, 184 mmol), DMF(1.00 g, 13.6 mmol) and methylene chloride (300 mL). The solution wascooled to 0° C. using an ice bath. Oxalyl chloride (28.0 g, 221 mmol)was added dropwise, and the reaction mixture was warmed to roomtemperature over 30 min and stirred for 5 h. After this time, theresulting solution was concentrated to afford a brown solid. This solidwas dissolved in anhydrous methanol (400 mL), and the solution wascooled to 0° C. Triethylamine (57 g, 552 mmol) was added to the reactionmixture, and it was stirred for a further 2 h at room temperature. Afterthis time, the reaction mixture was concentrated to dryness underreduced pressure. The residue was diluted with methylene chloride (300mL) and washed with water (200 mL) and saturated aqueous sodiumbicarbonate (200 mL). The organic layer was dried over sodium sulfate,filtered and concentrated under reduced pressure. The resulting residuewas titrated with hexane (200 mL) to afford 120a in 58% yield (19.1 g)as a white solid: mp 72-74° C.; ¹H NMR (300 MHz, DMSO-d₆) δ 7.13 (s,1H), 6.23 (s, 1H), 3.93 (t, 2H, J=6.0 Hz), 3.77 (s, 3H), 2.75 (t, 2H,J=6.0 Hz), 1.93 (m, 2H), 1.80 (m, 2H); (APCI+) m/z 180.1 (M+H)

Example 120b Methyl3-(Cyanomethyl)-5,6,7,8-tetrahydroindolizine-2-carboxylate 120b

A 500-mL three-neck round-bottomed flask equipped with an additionfunnel, thermometer and charged with 120a (6.70 g, 37.4 mmol),iodoacetonitrile (12.5 g, 74.9 mmol), iron (II) sulfate heptahydrate(5.20 g, 18.7 mmol) and dimethyl sulfoxide (250 mL). Hydrogen peroxide(35%, 18.2 g, 187 mmol) was added dropwise to the mixture over theperiod of 1 h through a syringe pump at room temperature using a waterbath. Iron (II) sulfate heptahydrate (2 to 3 equivalent) was added tothe reaction mixture in portions to keep the temperature between 25° C.to 35° C., until the color of the reaction mixture was deep red. WhenTLCs showed the reaction was not complete, more hydrogen peroxide (2-3equivalent) and more iron (II) sulfate heptahydrate (1-2 equivalents)were added in the same manner until the reaction was complete. Afterthat time, the reaction mixture was partitioned between saturated sodiumbicarbonate solution (200 mL) and ethyl acetate (400 mL). The organiclayer was separated, and the aqueous layer was extracted with ethylacetate (2×100 mL). The combined organic layers were washed withsaturated sodium thiosulfate solution (50 mL), dried over sodium sulfateand concentrated under reduced pressure. The residue was purified bycolumn chromatography to afford a 78% yield (6.40 g) of 120b as a yellowoil: ¹H NMR (500 MHz, CDCl₃) δ 6.23 (s, 1H), 4.23 (s, 2H), 3.94 (t, 2H,J=6.5 Hz), 3.81 (s, 3H), 2.74 (t, 2H, J=6.5 Hz), 2.00 (m, 2H), 1.83 (m,2H); (APCI+) m/z 219.3 (M+H)

Example 120c Methyl3-(2-Aminoethyl)-5,6,7,8-tetrahydroindolizine-2-carboxylate HydrogenChloride Salt 120c

Methyl 3-(Cyanomethyl)-5,6,7,8-tetrahydroindolizine-2-carboxylate 120bwas hydrogenated with platinum oxide catalyst under 50 psi of hydrogenin ethanol and ethyl acetate in the presence of hydrogen chlorideovernight at room temperature to give 120c (380 mg, 1.74 mmol) which wasused in the next step.

Example 120d 3,4,6,7,8,9-Hexahydropyrido[3,4-b]indolizin-1(2H)-one 120d

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and nitrogen inlet was purged with nitrogen and charged with120c (prepared above, estimated 1.74 mmol, presuming quantitativeyield), sodium ethoxide (354 mg, 5.22 mmol) and ethanol (20 mL). Themixture was stirred at 55° C. for 5 h. After that time, the reactionmixture was concentrated under reduced pressure and the residue waspartitioned between ethyl acetate (200 mL) and water (100 mL). Theorganic layer was separated, and the aqueous layer was extracted withethyl acetate (2×100 mL). The combined organic layers were washed withbrine, dried over sodium sulfate and concentrated under reducedpressure. The residue was purified by column chromatography to afford a67% yield (220 mg) of 120d as a white solid: mp 195-197° C.; ¹H NMR (500MHz, DMSO-d₆) δ 6.76 (s, 1H), 5.89 (s, 1H), 3.78 (t, 2H, J=6.5 Hz), 3.35(m, 2H), 2.66 (m, 4H), 1.87 (m, 2H), 1.72 (m, 2H); (APCI+) m/z 191.3(M+H)

Example 120e2-Bromo-4-fluoro-6-(1-oxo-3,4,6,7,8,9-hexahydropyrido[3,4-b]-indolizin-2(1H)-yl)benzylAcetate 120e

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and reflux condenser was charged with 1,4-dioxane (60 mL),2,6-dibromo-4-fluorobenzyl acetate (1.9 g, 6.0 mmol), 120d (400 mg, 2.0mmol), and cesium carbonate (1.3 g, 4.0 mmol). After bubbling nitrogenthrough the resulting mixture for 30 minutes, Xantphos (120 mg, 0.2mmol) and tris(dibenzylideneacetone)dipalladium(0) (180 mg, 0.2 mmol)were added, and the reaction mixture was heated at 100° C. for 12 h.After this time the reaction was cooled to room temperature, partitionedbetween ethyl acetate (40 mL) and water (40 mL), and filtered. Theaqueous layer was separated and extracted with ethyl acetate (3×70 mL).The combined organic layer was washed with brine (30 mL) and dried oversodium sulfate. The drying agent was removed by filtration and thefiltrate was concentrated under reduced pressure. The residue waspurified on flush column eluting with 2:1 petroleum ether/ethyl acetateto afford 120e (421 mg, 46%) as yellow solid. MS: [M+H]⁺ 435. ¹H NMR(500 MHz, MeOD) δ 7.52-7.50 (m, 1H), 7.23-7.20 (m, 1H), 6.14 (s, 1H),5.20-5.10 (m, 2H), 4.09-4.06 (m, 1H), 3.95-3.92 (m, 1H), 3.88-3.85 (m,1H), 3.78-3.75 (m, 1H), 3.07-2.97 (m, 2H), 2.81-2.77 (m, 2H), 2.03-2.00(m, 5H), 1.87-1.84 (m, 2H).

Example 120f4-Fluoro-2-(1-oxo-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-2(1H)-yl)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylAcetate 120f

A 100-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 1,4-dioxane (30 mL),120e (1000 mg. 2.30 mmol), bis(pinacolato)diboron (3.03 g, 11.5 mmol),Pd(dppf)Cl₂ (94 mg, 0.11 mmol), and potassium acetate (676 mg, 6.90mmol). After bubbling nitrogen through the mixture for 10 minutes, itwas heated at 90° C. for 3 h. Then it was filtered and the filtrate wasevaporated in vacuo to afford 120f (1.2 g, 108%) as black oil. MS-ESI:[M+H]⁺ 483.2

Example 120g4-Fluoro-2-(6-(4-(4-(oxetan-3-yl)piperazin-1-yl)phenylamino)-9-((2-(trimethylsilyl)ethoxy)methyl)-9H-purin-2-yl)-6-(1-oxo-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-2(1H)-yl)benzylAcetate 120g

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 120f (400 mg, 0.82mmol),2-iodo-N-(4-(4-(oxetan-3-yl)piperazin-1-yl)phenyl)-9-((2-(trimethylsilyl)ethoxy)methyl)-9H-purin-6-amine117f (604 mg, 0.99 mmol), PdCl₂(dppf) (33 mg, 0.040 mmol), K₃PO₄ (347mg, 1.64 mmol), and sodium acetate (134 mg, 1.64 mmol), acetonitrile (15mL), and water (1 mL). The system was evacuated and refilled with N₂.The reaction mixture was heated at 100° C. for 2 h. It was then cooledto room temperature and filtered. The filtrate was concentrated underreduced pressure and the resulting residue was purified by silica-gelcolumn chromatography eluting with 30:1 dichloromethane/methanol toafford 120g (460 mg, 66%) as a green solid. MS-ESI: [M+H]⁺ 836.4.

Example 120h4-Fluoro-2-(6-(4-(4-(oxetan-3-yl)piperazin-1-yl)phenylamino)-9H-purin-2-yl)-6-oxo-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-2(1H)-yl)benzylAcetate 120h

A 50-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with 120g (350 mg, 0.42mmol), TBAF (925 mg, 2.93 mmol), and THF (10 mL). The reaction mixturewas heated at 80° C. for 17 h. It was then cooled to room temperatureand filtered. The filtrate was concentrated under reduced pressure andthe resulting residue was purified by silica-gel column chromatographyeluting with 30:1 dichloromethane/methanol to afford 120h (240 mg, 83%)as a yellow solid. MS-ESI: [M+H]⁺ 706.3.

Example 1202-[5-fluoro-2-(hydroxymethyl)-3-[6-[4-[4-(oxetan-3-yl)piperazin-1-yl]anilino]-9H-purin-2-yl]phenyl]-3,4,6,7,8,9-hexahydropyrido[3,4-b]indolizin-1-one120

To a solution of 120h (240 mg, 0.34 mmol) in propan-2-ol (8 mL),tetrahydrofuran (8 mL), and water (1.5 mL) was added lithium hydroxide(25 mg, 1.02 mmol). The mixture was stirred at 30° C. for 1.5 h. It wasevaporated and the residue was purified by reverse-phase prep-HPLC toafford 120 (116 mg, 51%) as a yellow solid. MS-ESI: [M+H]⁺ 664.3. ¹H NMR(500 MHz, CDCl₃) δ 8.29 (s, 1H), 7.81 (d, J=8.0 Hz, 1H), 7.69 (d, J=8.0Hz, 2H), 7.08 (d, J=6.5 Hz, 1H), 6.91 (d, J=8.0 Hz, 2H), 6.29 (s, 1H),4.71-4.57 (m, 6H), 4.05-4.03 (m, 1H), 3.86-3.77 (m, 3H), 3.63-3.61 (m,1H), 3.23-3.22 (m, 4H), 3.05-3.03 (m, 1H), 3.89-2.79 (m, 3H), 2.54-2.56(m, 4H), 2.00-1.98 (m, 2H), 1.84-1.82 (m, 2H).

Example 121a6-Chloro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)imidazo[1,2-a]pyridin-8-amine121a

A 50-mL round-bottomed flask equipped with a reflux condenser wascharged with 8-bromo-6-chloroimidazo[1,2-a]pyridine 101a (264 mg, 1.14mmol), 5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-amine (328 mg, 1.14mmol), Pd₂(dba)₃ (102 mg, 0.11 mmol), Xantphos (63 mg, 0.11 mmol),Cs₂CO₃ (3.58 g, 11.0 mmol), dioxane (20 mL). After three cycles ofvacuum/argon flush, the mixture was heated at 100° C. overnight. It wasthen filtered and the filtrate was evaporated under reduced pressure.The residue was purified by silica-gel column chromatography elutingwith 1:50 methanol/dichloromethane to afford 121a as an orange solid(290 mg, 66%). MS-ESI: [M+H]⁺ 385.1

Example 1212-[3-(hydroxymethyl)-4-[8-[[5-[4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]imidazo[1,2-a]pyridin-6-yl]-2-pyridyl]-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1-one121

A microwave vial was charged with 121a (100 mg, 0.26 mmol),3-(acetoxymethyl)-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)pyridin-4-ylboronicacid 114e (150 mg, 0.39 mmol), Pd₂(dba)₃ (23 mg, 0.025 mmol), P(Cy)₃ (7mg, 0.025 mmol), Cs₂CO₃ (170 mg, 0.52 mmol), dioxane (5 mL), and water(0.1 mL). After three cycles of vacuum/argon flush, the reaction mixturewas stirred at 130° C. under microwave irradiation for 1 h. It was thenfiltered and the filtrate was evaporated under reduced pressure. Theresidue was purified with reverse-phase prep-HPLC to afford 121 (66 mg,39%). MS-ESI: [M+H]⁺ 646.3. ¹H NMR (500 MHz, DMSO-d₆) δ 8.99 (s, 1H),8.53 (d, J=5.0 Hz, 1H), 8.31 (d, J=1.5 Hz, 1H), 8.23 (d, J=1.5 Hz, 1H),7.99 (d, J=1.0 Hz, 1H), 7.90 (d, J=3.0 Hz, 1H), 7.58 (d, J=1.5 Hz, 1H),7.43-7.40 (m, 2H), 7.35 (d, J=9.0 Hz, 1H), 6.58 (s, 1H), 4.95 (bs, 1H),4.56 (t, J=6.5 Hz, 2H), 4.46 (t, J=6.5 Hz, 2H), 4.42-4.40 (m, 2H),4.28-4.09 (m, 3H), 3.94-3.87 (m, 1H), 3.45-3.42 (m, 1H), 3.10-3.08 (m,4H), 2.66-2.56 (m, 2H), 2.50-2.47 (m, underneath solvent peak, 2H),2.40-2.38 (m, 4H), 1.80-1.69 (m, 4H).

Example 122a(4-(8-(5-(4-(Oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)[1,2,4]triazolo[1,5-a]pyridin-6-yl)-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)pyridin-3-yl)methylAcetate 122a

A sealed tube was charged with6-chloro-N-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-yl)[1,2,4]triazolo[1,5-a]pyridin-8-amine108d (196 mg, 0.51 mmol),3-(acetoxymethyl)-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)pyridin-4-ylboronicacid 114e (200 mg, 0.52 mmol), PdCl₂(dppf) (24 mg, 0.030 mmol), K₃PO₄(212 mg, 1.0 mmol), sodium acetate (83 mg, 1.0 mmol), acetonitrile (10mL), and water (0.2 mL). The mixture was heated at 140° C. for 0.5 h. Itwas then cooled to room temperature and filtered. The filtrate wasevaporated in vacuo. The residue was purified by silica-gel columnchromatography eluting with 20:1 dichloromethane/methanol to afford 122a(200 mg, 56%) as a white solid. MS-ESI: [M+H]⁺ 689.3.

Example 1222-[3-(hydroxymethyl)-4-[8-[[5-[4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-[1,2,4]triazolo[1,5-a]pyridin-6-yl]-2-pyridyl]-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1-one122

A mixture of 122a (200 mg, 0.29 mmol) and lithium hydroxide (42 mg, 1.0mmol) in i-propanol/THF (1:1, 3.5 mL) and water (1 mL) was stirred at40° C. for 0.5 h. The mixture was evaporated under reduced pressure andthe residue was diluted with water (5 mL). It was extracted with ethylacetate (2×5 mL). The combined ethyl acetate extract was concentratedunder reduced pressure and the residue was purified by reverse-phaseprep-HPLC to afford 122 (70 mg, 38%) as pale yellow solid. MS-ESI:[M+H]⁺ 647.3. ¹H NMR (500 MHz, DMSO-d₆) δ 9.36 (s, 1H), 8.69 (s, 1H),8.61 (s, 1H), 8.57-8.55 (m, 2H), 7.93 (d, J=2.5 Hz, 1H), 7.48-7.43 (m,2H), 7.37-7.35 (m, 1H), 6.59 (s, 1H), 5.05 (t, J=5.0 Hz, 1H), 4.57-4.55(m, 2H), 4.61-4.59 (m, 2H), 4.40-4.39 (m, 2H), 4.26-4.12 (m, 3H),3.92-3.89 (m, 1H), 3.43 (t, J=6.0 Hz, 1H), 3.11-3.09 (m, 4H), 2.63-2.57(m, 2H), 2.50-2.47 (m, 2H), 2.40-2.39 (m, 4H), 1.79-1.77 (m, 2H),1.70-1.68 (m, 2H).

Example 123a(2-{4,4-Dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}-4-fluoro-6-[6-({4-[4-(oxetan-3-yl)piperazin-1-yl]phenyl}amino)-9-{[2-(trimethylsilyl)ethoxy]methyl}purin-2-yl]phenyl)methylAcetate 123a

A 25-mL single-neck round-bottomed flask equipped with a magneticstirrer and a reflux condenser was charged with2-iodo-N-(4-(4-(oxetan-3-yl)piperazin-1-yl)phenyl)-9-((2-(trimethylsilyl)ethoxy)methyl)-9H-purin-6-amine117f (360 mg, 0.60 mmol),{2-[(acetyloxy)methyl]-3-{4,4-dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}-5-fluorophenyl}boronicacid 110g (360 mg, 0.90 mmol), Pd(dppf)Cl₂ (30 mg, 0.030 mmol), K₃PO₄(270 mg, 1.2 mmol), sodium acetate (180 mg, 1.2 mmol), water (0.5 mL),and acetonitrile (10 mL). After three cycles of vacuum/argon flush, themixture was heated at 100° C. for 2 h. It was then cooled to roomtemperature and filtered. The filtrate was concentrated under reducedpressure and the resulting residue was purified by silica-gel columnchromatography eluting with 20:1 dichloromethane/methanol to afford 123aas a yellow solid (200 mg, 35%). MS-ESI: [M+H]⁺ 850.4

Example 123b(2-{4,4-Dimethyl-9-oxo-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-10-yl}-4-fluoro-6-[6-({4-[4-(oxetan-3-yl)piperazin-1-yl]phenyl}amino)-9H-purin-2-yl]phenyl)methylAcetate 123b

A mixture of 123a (200 mg, 0.25 mmol) and CF₃COOH (4 mL) was stirred atroom temperature for 2 h. The mixture was then concentrated underreduced pressure to afford crude 123b as a yellow solid (160 mg, 90%),which was used for the next step without further purification. MS-ESI:[M+H]⁺ 720.4

Example 1233-[5-fluoro-2-(hydroxymethyl)-3-[6-[4-[4-(oxetan-3-yl)piperazin-1-yl]anilino]-9H-purin-2-yl]phenyl]-7,7-dimethyl-1,2,6,8-tetrahydrocyclopenta[3,4]pyrrolo[3,5-b]pyrazin-4-one123

The mixture of 123b (160 mg, 0.20 mmol) and lithium hydroxide (48 mg,2.0 mmol) in THF/i-propanol (5:3, 8 mL) and water (2 mL) was stirred at30° C. for 1 h. The mixture was evaporated in vacuo and the residue wasdiluted with water (5 mL). It was then extracted with ethyl acetate(2×10 mL). The combined ethyl acetate extract was concentrated underreduced pressure and the residue was purified by reverse-phase prep-HPLCto afford 123 (40 mg, 32%) as a white solid. MS-ESI: [M+H]⁺ 678.3. ¹HNMR (500 MHz, CDCl₃) δ 12.28 (s, 1H), 8.62 (s, 1H), 7.89-7.87 (m, 1H),7.72-7.67 (m, 3H), 7.13-7.11 (m, 1H), 6.99-6.97 (m, 2H), 6.85 (s, 1H),4.73-4.65 (m, 6H), 4.25-4.21 (m, 1H), 4.16-4.09 (m, 2H), 3.89-3.87 (m,1H), 3.59-3.57 (m, 1H), 3.25-3.23 (m, 4H), 2.56-2.51 (m, overlap, 8H),1.28 (s, 3H), 1.27 (s, 3H).

Example 124a (3-Nitro-1H-pyrazol-5-yl)methanol 124a

A 3-L three-neck round-bottomed flask equipped with a mechanicalstirrer, addition funnel and nitrogen inlet was purged with nitrogen andcharged with 3-nitropyrazole-5-carboxylic acid (28.0 g, 178 mmol) andTHF (420 mL) and cooled to −5° C. using an ice/acetone bath. Borane-THFcomplex solution (1.0 M, 535 mL, 535 mmol) was added at a rate thatmaintained the internal reaction temperature below 5° C. After theaddition was complete the cooling bath was removed and the reaction wasstirred at room temperature for 18 h. After this time the reaction wascooled to −5° C. using an ice/acetone bath, water (70 mL) and 4Nhydrochloric acid (70 mL) was added and the reaction was stirred atreflux for 1 h in order to destroy the borane complex with pyrazole. Thereaction was cooled to room temperature and concentrated under reducedpressure to a volume of approximately 30 mL. Ethyl acetate (175 mL) wasadded and the mixture stirred for 15 min. The aqueous layer wasseparated and extracted with ethyl acetate (4×200 mL). The combinedorganic layers were washed with saturated aqueous sodium bicarbonate(2×50 mL), brine (50 mL) and dried over sodium sulfate, the drying agentwas removed by filtration, and the filtrate concentrated under reducedpressure to afford (3-nitro-1H-pyrazol-5-yl)methanol 124a in a 94% yield(24.0 g) as a light yellow solid: ¹H NMR (300 MHz, DMSO-d₆) δ 13.90 (brs, 1H), 6.87 (s, 1H), 5.58 (t, 1H, J=5.4 Hz), 4.53 (d, 2H, J=5.1 Hz); MS(ESI+) m/z 144.0 (M+H)

Example 124b (1-(2-Bromoethyl)-3-nitro-1H-pyrazol-5-yl)methanol 124b

A 1-L three-necked round-bottomed flask equipped with a mechanicalstirrer and thermoregulator was purged with nitrogen and charged with124a (25.0 g, 175 mmol), DMF (250 mL), and cesium carbonate (70.0 g, 215mmol) was heated at 104° C. for 5 min. The reaction mixture was thencooled to 0° C. using an ice/acetone bath and dibromoethane (329 g, 1.75mol) was added portionwise (no exotherm). The reaction was stirred at 0°C. for 1 then at room temperature for 4 h. After this time a solution ofKH₂PO4 (40 g) in water (400 mL) was added slowly. The reaction mixturestirred at room temperature for 30 min. Ethyl acetate (450 mL) was addedand the aqueous layer was separated and extracted with ethyl acetate(2×100 mL). The combined organic layers were washed with water (200 mL),brine (200 mL), dried over sodium sulfate, and the drying agent wasremoved by filtration. The filtrate was concentrated under reducedpressure to afford an 86% yield (37.5 g) of crude 124b as an orange oil:¹H NMR (300 MHz, CDCl₃) δ 6.85 (s, 1H), 4.82 (d, 2H, J=5.4 Hz), 4.66 (t,2H, J=6.3 Hz), 3.83 (t, 2H, J=6.3 Hz); MS (ESI+) m/z 249.9 (M+H).

Example 124c 1-(2-Bromoethyl)-5-(bromomethyl)-3-nitro-1H-pyrazole 124c

A 500-mL three-necked round-bottomed flask equipped with a magneticstirrer, nitrogen inlet and reflux condenser was purged with nitrogenand charged with 124b (37.0 g, 148 mmol) and chloroform (160 mL). Thereaction was cooled to −5° C. using an ice/acetone bath and phosphoroustribromide (40.0 g, 148 mmol) was added portionwise. The cooling bathwas removed and the reaction stirred at reflux for 2 h. After this time,the reaction was cooled to −5° C. and saturated aqueous sodiumbicarbonate (250 mL) was added until a pH of 8.5 was reached. Themixture was extracted with ethyl acetate (3×150 mL) and the combinedorganic layers were washed with saturated aqueous sodium carbonate (2×50mL), brine (75 mL), dried over sodium sulfate and the drying agent wasremoved by filtration. The filtrate was concentrated under reducedpressure to afford a yellow residue that was dissolved with gentleheating in methylene chloride (60 mL). Hexanes (approximately 20 mL) wasadded and the solution became cloudy. The mixture was heated until asolid precipitate formed, methylene chloride (9 mL) was added and thesolution became clear. The solution was left to cool to room temperatureand after 4 h the resulting crystals were collected by vacuumfiltration. The filter cake was washed with an ice-cold 1:2 mixture ofmethylene chloride:hexanes (2×20 mL) to afford1-(2-bromoethyl)-5-(bromomethyl)-3-nitro-1H-pyrazole (19.7 g). Thecombined filtrates were evaporated and the procedure was performed againto afford an additional 9.70 g of1-(2-bromoethyl)-5-(bromo-methyl)-3-nitro-1H-pyrazole. The solids werecombined and dried under high vacuum for 18 h to afford a 57% yield(26.0 g) of 1-(2-bromoethyl)-5-(bromomethyl)-3-nitro-1H-pyrazole 124c aswhite crystals: mp 95-97° C.; ¹H NMR (300 MHz, CDCl₃) δ 6.93 (s, 1H),4.63 (t, 2H, J=6.0 Hz), 4.54 (s, 2H), 3.86 (t, 2H, J=6.0 Hz).

Example 124d 5-Methyl-2-nitro-4,5,6,7-tetrahydropyrazolo[1,5-c]pyrazine124d

A 1-L single-neck round-bottomed flask equipped with a magnetic stirrerand nitrogen inlet was charged with THF (350 mL), 124c (10.0 g, 32.2mmol), 2M methylamine solution in THF (113 mL, 225 mmol) and stirred atroom temperature for 72 h. After this time the reaction was concentratedto dryness under reduced pressure, and the resulting solid was stirredwith a mixture of ethyl acetate (75 mL) and 10% aqueous potassiumcarbonate (75 mL). The aqueous layer was separated and extracted withethyl acetate (2×75 mL). The combined organic extracts were washed with10% aqueous potassium carbonate (75 mL), followed by brine (50 mL) anddried over sodium sulfate. The drying agent was removed by filtration,and the filtrate concentrated under reduced pressure to afford 124d in97% yield (5.70 g) as a yellow solid: ¹H NMR (300 MHz, CDCl₃) δ 6.62 (s,1H), 4.28 (t, 2H, J=5.4 Hz), 3.67 (s, 2H), 2.95 (t, 2H, J=5.4 Hz), 2.52(s, 3H); MS (ESI+) m/z 183.0 (M+H)

Example 124e 5-Methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-amine124e

A 500-mL Parr reactor bottle was purged with nitrogen and charged with10% palladium on carbon (50% wet, 800 mg dry weight) and a solution of124d (4.00 g, 2.20 mmol) in ethanol (160 mL). The bottle was attached toParr hydrogenator, evacuated, charged with hydrogen gas to a pressure of45 psi and shaken for 2 h. After this time, the hydrogen was evacuated,and nitrogen was charged into the bottle. Celite 521 (1.0 g) was added,and the mixture was filtered through a pad of Celite 521. The filtercake was washed with ethanol (2×75 mL), and the combined filtrates wereconcentrated to dryness under reduced pressure to afford a 99% yield of124e (3.31 g) as an orange solid: ¹H NMR (300 MHz, CDCl₃) δ 5.34 (s,1H), 3.98 (t, 2H, J=5.4 Hz), 3.52 (s, 3H), 2.84 (t, 2H, J=5.7 Hz), 2.45(s, 3H); MS (ESI+) m/z 153.1 (M+H)

Example 124f6-Chloro-N-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)imidazo[1,2-b]pyridazin-8-amine124f

Following the procedures as described in Example 104b, and starting with8-bromo-6-chloroimidazo[1,2-b]pyridazine 104a, 1.43 g, 6.2 mmol) and124e (900 mg, 5.9 mmol) afforded 124f was as a yellow solid (800 mg,44%). MS-ESI: [M+H]⁺ 304.1

Example 124g6-Bromo-N-(5-methyl-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrazin-2-yl)imidazo[1,2-b]pyridazin-8-amine124g

To an autoclave was charged 124f (800 mg, 2.6 mmol)) and HBr/AcOH (60mL). It was heated at 150° C. for 18 h. The reaction mixture wasconcentrated under reduced pressure to give black oil. The oil wasrecrystallized from methanol (30 mL)/dichloromethane (30 mL)/petroleumether (90 mL) to 124g as a yellow solid (600 mg, 66%). MS-ESI: [M+H]⁺348.1

Example 1242-[3-(hydroxymethyl)-4-[8-[(5-methyl-6,7-dihydro-4H-pyrazolo[1,5-a]pyrazin-2-yl)amino]imidazo[1,2-b]pyridazin-6-yl]-2-pyridyl]-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1-one124

Following the procedure described in Example 123, and starting with 124g(900 mg, 2.60 mmol) and3-(acetoxymethyl)-2-(1-oxo-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-2(1H)-yl)pyridin-4-ylboronicacid 114e (1.01 g, 2.6 mmol), 124 was obtained as a white solid (147 mg,10%). MS-ESI: [M+H]⁺ 565.3. ¹H NMR (500 MHz, DMSO-d₆) δ 10.02 (s, 1H),8.56 (d, J=5.0 Hz, 1H), 8.17 (s, 1H), 7.75 (s, 1H), 7.66 (s, 1H), 7.46(d, J=5.0 Hz, 1H), 6.58 (s, 1H), 6.03 (s, 1H), 4.73 (t, J=5.0 Hz, 1H),4.62-4.58 (m, 1H), 4.40-4.37 (m, 1H), 4.27-4.19 (m, 2H), 4.10-4.08 (m,1H), 4.00-3.93 (m, 3H), 3.54 (s, 2H), 2.82 (t, J=5.0 Hz, 2H), 2.64-2.56(m, 2H), 2.47-2.46 (m, 2H), 2.37 (s, 3H), 1.79-1.68 (m, 4H).

Example 901 Biochemical Btk Assay

A generalized procedure for a standard biochemical Btk Kinase Assay thatcan be used to test Formula I compounds is as follows. A master mixminus Btk enzyme is prepared containing 1X Cell Signaling kinase buffer(25 mM Tris-HCl, pH 7.5, 5 mM beta-glycerophosphate, 2 mMdithiothreitol, 0.1 mM Na₃VO₄, 10 mM MgCl₂), 0.5 μM Promega PTKBiotinylated peptide substrate 2, and 0.01% BSA. A master mix plus Btkenzyme is prepared containing 1X Cell Signaling kinase buffer, 0.5 μMPTK Biotinylated peptide substrate 2, 0.01% BSA, and 100 ng/well (0.06mU/well) Btk enzyme. Btk enzyme is prepared as follows: full lengthhuman wildtype Btk (accession number NM-000061) with a C-terminal V5 and6×His tag was subcloned into pFastBac vector for making baculoviruscarrying this epitope-tagged Btk. Generation of baculovirus is donebased on Invitrogen's instructions detailed in its published protocol“Bac-to-Bac Baculovirus Expression Systems” (Cat. Nos. 10359-016 and10608-016). Passage 3 virus is used to infect Sf9 cells to overexpressthe recombinant Btk protein. The Btk protein is then purified tohomogeneity using Ni-NTA column. The purity of the final proteinpreparation is greater than 95% based on the sensitive Sypro-Rubystaining A solution of 200 μM ATP is prepared in water and adjusted topH7.4 with 1N NaOH. A quantity of 1.25 μL of compounds in 5% DMSO istransferred to a 96-well ½ area Costar polystyrene plate. Compounds aretested singly and with an 11-point dose-responsive curve (startingconcentration is 10 μM; 1:2 dilution). A quantity of 18.75 μL of mastermix minus enzyme (as a negative control) and master mix plus enzyme istransferred to appropriate wells in 96-well ½ area costar polystyreneplate. 5 μL of 200 μM ATP is added to that mixture in the 96-well ½ areaCostar polystyrene plate for final ATP concentration of 40 μM. Thereaction is allowed to incubate for 1 hour at room temperature. Thereaction is stopped with Perkin Elmer 1X detection buffer containing 30mM EDTA, 20 nM SA-APC, and 1 nM PT66 Ab. The plate is read usingtime-resolved fluorescence with a Perkin Elmer Envision using excitationfilter 330 nm, emission filter 665 nm, and 2^(nd) emission filter 615nm. IC₅₀ values are subsequently calculated. Alternatively, theLanthascreen assay can be used to evaluate Btk activity throughquantification of its phosphorylated peptide product. The FRET(Fluorescence Resonance Energy Transfer) that occurs between thefluorescein on the peptide product and the terbium on the detectionantibody decreases with the addition of inhibitors of Btk that reducethe phosphorylation of the peptide. In a final reaction volume of 25 uL,Btk (h) (0.1 ng/25 ul reaction) is incubated with 50 mM Hepes pH 7.5, 10mM MgCl₂, 2 mM MnCl₂, 2 mM DTT, 0.2 mM NaVO4, 0.01% BSA, and 0.4 uMfluorescein poly-GAT. The reaction is initiated by the addition of ATPto 25 uM (Km of ATP). After incubation for 60 minutes at roomtemperature, the reaction is stopped by the addition of a finalconcentration of 2 nM Tb-PY20 detection antibody in 60 mM EDTA for 30minutes at room temperature. Detection is determined on a Perkin ElmerEnvision with 340 nM excitation and emission at 495 nm and 520 nm.Exemplary Btk inhibition IC70 values are in Tables 1 and 2.

Example 902 Ramos Cell Btk Assay

Another generalized procedure for a standard cellular Btk Kinase Assaythat can be used to test Formula I compounds is as follows. Ramos cellsare incubated at a density of 0.5×10⁷ cells/ml in the presence of testcompound for 1 hr at 37° C. Cells are then stimulated by incubating with10 μg/ml anti-human IgM F(ab)₂ for 5 minutes at 37° C. Cells arepelleted, lysed, and a protein assay is performed on the cleared lysate.Equal protein amounts of each sample are subject to SDS-PAGE and westernblotting with either anti-phosphoBtk(Tyr223) antibody (Cell SignalingTechnology #3531; Epitomics, cat. #2207-1) or phosphoBtk(Tyr551)antibody (BD Transduction Labs #558034) to assess Btkautophosphorylation or an anti-Btk antibody (BD Transduction Labs#611116) to control for total amounts of Btk in each lysate.

Example 903 B-Cell Proliferation Assay

A generalized procedure for a standard cellular B-cell proliferationassay that can be used to test Formula I compounds is as follows.B-cells are purified from spleens of 8-16 week old Balb/c mice using aB-cell isolation kit (Miltenyi Biotech, Cat #130-090-862). Testingcompounds are diluted in 0.25% DMSO and incubated with 2.5×10⁵ purifiedmouse splenic B-cells for 30 min prior to addition of 10 μg/ml of ananti-mouse IgM antibody (Southern Biotechnology Associates Cat #1022-01)in a final volume of 100 μl. Following 24 hr incubation, 1 μCi³H-thymidine is added and plates are incubated an additional 36 hr priorto harvest using the manufacturer's protocol for SPA[³H]thymidine uptakeassay system (Amersham Biosciences # RPNQ 0130). SPA-bead basedfluorescence is counted in a microbeta counter (Wallace Triplex 1450,Perkin Elmer).

Example 904 T Cell Proliferation Assay

A generalized procedure for a standard T cell proliferation assay thatcan be used to test Formula I compounds is as follows. T cells arepurified from spleens of 8-16 week old Balb/c mice using a Pan T cellisolation kit (Miltenyi Biotech, Cat #130-090-861). Testing compoundsare diluted in 0.25% DMSO and incubated with 2.5×10⁵ purified mousesplenic T cells in a final volume of 100 μl in flat clear bottom platesprecoated for 90 min at 37° C. with 10 μg/ml each of anti-CD3 (BD#553057) and anti-CD28 (BD #553294) antibodies. Following 24 hrincubation, 1 μCi ³H-thymidine is added and plates incubated anadditional 36 hr prior to harvest using the manufacturer's protocol forSPA[³H] thymidine uptake assay system (Amersham Biosciences # RPNQ0130). SPA-bead based fluorescence was counted in a microbeta counter(Wallace Triplex 1450, Perkin Elmer).

Example 905 CD86 Inhibition Assay

A generalized procedure for a standard assay for the inhibition of Bcell activity that can be used to test Formula I compounds is asfollows. Total mouse splenocytes are purified from spleens of 8-16 weekold Balb/c mice by red blood cell lysis (BD Pharmingen #555899). Testingcompounds are diluted to 0.5% DMSO and incubated with 1.25×10⁶splenocytes in a final volume of 200 μl in flat clear bottom plates(Falcon 353072) for 60 min at 37° C. Cells are then stimulated with theaddition of 15 μg/ml IgM (Jackson ImmunoResearch 115-006-020), andincubated for 24 hr at 37° C., 5% CO₂. Following the 24 hr incubation,cells are transferred to conical bottom clear 96-well plates andpelleted by centrifugation at 1200×g×5 min. Cells are preblocked byCD16/CD32 (BD Pharmingen #553142), followed by triple staining withCD19-FITC (BD Pharmingen #553785), CD86-PE (BD Pharmingen #553692), and7AAD (BD Pharmingen #51-68981E). Cells are sorted on a BD FACSCaliburand gated on the CD19⁺/7AAD⁻ population. The levels of CD86 surfaceexpression on the gated population is measured versus test compoundconcentration.

Example 906 B-ALL Cell Survival Assay

The following is a procedure for a standard B-ALL (acute lymphoblasticleukemia) cell survival study using an XTT readout to measure the numberof viable cells. This assay can be used to test Formula I compounds fortheir ability to inhibit the survival of B-ALL cells in culture. Onehuman B-cell acute lymphoblastic leukemia line that can be used isSUP-B15, a human Pre-B-cell ALL line that is available from the ATCC.

SUP-B15 pre-B-ALL cells are plated in multiple 96-well microtiter platesin 100 μA of Iscove's media+20% FBS at a concentration of 5×10⁵cells/ml. Test compounds are then added with a final conc. of 0.4% DMSO.Cells are incubated at 37° C. with 5% CO₂ for up to 3 days. After 3 dayscells are split 1:3 into fresh 96-well plates containing the testcompound and allowed to grow up to an additional 3 days. After each 24 hperiod, 50 ul of an XTT solution is added to one of the replicate96-well plates and absorbance readings are taken at 2, 4 and 20 hoursfollowing manufacturer's directions. The reading taken with an OD forDMSO only treated cells within the linear range of the assay (0.5-1.5)is then taken and the percentage of viable cells in the compound treatedwells are measured versus the DMSO only treated cells.

Example 907 CD69 Whole Blood Assay

Human blood is obtained from healthy volunteers, with the followingrestrictions: 1 week drug-free, non-smokers. Blood (approximately 20 mlsto test 8 compounds) is collected by venipuncture into Vacutainer®(Becton, Dickinson and Co.) tubes with sodium heparin.

Solutions of Formula I compounds at 10 mM in DMSO are diluted 1:10 in100% DMSO, then are diluted by three-fold serial dilutions in 100% DMSOfor a ten point dose-response curve. The compounds are further diluted1:10 in PBS and then an aliquot of 5.5 μl of each compound is added induplicate to a 2 ml 96-well plate; 5.5 μl of 10% DMSO in PBS is added ascontrol and no-stimulus wells. Human whole blood—HWB (100 μl) is addedto each well. After mixing the plates are incubated at 37° C., 5% CO₂,100% humidity for 30 minutes. Goat F(ab′)₂ anti-human IgM (10 μl of a500 μg/ml solution, 50 μg/ml final) is added to each well (except theno-stimulus wells) with mixing and the plates are incubated for anadditional 20 hours. At the end of the 20 hour incubation, samples areincubated with fluorescent labeled antibodies for 30 minutes, at 37° C.,5% CO₂, 100% humidity. Include induced control, unstained and singlestains for compensation adjustments and initial voltage settings.Samples are then lysed with PharM Lyse™ (BD Biosciences Pharmingen)according to the manufacturer's instructions. Samples are thentransferred to a 96 well plate suitable to be run on the BD BiosciencesHTS 96 well system on the LSRII machine. Data acquired and MeanFluorescence Intensity values were obtained using BD Biosciences DIVASoftware. Results are initially analyzed by FACS analysis software (FlowJo). The inhibitory concentrations (IC50, IC70, IC90, etc.) for testcompounds is defined as the concentration which decreases by, forexample 50%, the percent positive of CD69 cells that are also CD20positive stimulated by anti-IgM (average of 8 control wells, aftersubtraction of the average of 8 wells for the no-stimulus background).The IC70 values are calculated by Prism version 5, using a nonlinearregression curve fit and are shown in Tables 1 and 2.

Example 908 In Vitro Cell Proliferation Assay

Efficacy of Formula I compounds are measured by a cell proliferationassay employing the following protocol (Mendoza et al (2002) Cancer Res.62:5485-5488). The CellTiter-Glo® Luminescent Cell Viability Assay,including reagents and protocol are commercially available (PromegaCorp., Madison, Wis., Technical Bulletin TB288). The assay assesses theability of compounds to enter cells and inhibit cell proliferation. Theassay principle is based on the determination of the number of viablecells present by quantitating the ATP present in a homogenous assaywhere addition of the Cell-Titer Glo reagent results in cell lysis andgeneration of a luminescent signal through the luciferase reaction. Theluminescent signal is proportional to the amount of ATP present.

A panel of B-cell lymphoma cell lines (BJAB, SUDHL-4, TMD8, OCI-Ly10,OCI-Ly3, WSU-DLCL2) are plated into 384-well plate in normal growthmedium, and serially diluted BTK inhibitors or DMSO alone were added toeach well. Cell viability is assessed after 96 hour incubation byCellTiter-Glo® (Promega). Data may be presented as Relative cellviability in BTK inhibitor-treated cells relative to DMSO-treatedcontrol cells. Data points are the mean of 4 replicates at each doselevel. Error bars represent SD from the mean.

Procedure: Day 1—Seed Cell Plates (384-well black, clear bottom,microclear, TC plates with lid from Falcon #353962), Harvest cells, Seedcells at 1000 cells per 54 μl per well into 384 well Cell Plates for 3days assay. Cell Culture Medium: RPMI or DMEM high glucose, 10% FetalBovine Serum, 2 mM L-Glutamine, P/S. Incubate 0/N at 37° C., 5% CO2.

Day 2—Add Drug to Cells, Compound Dilution, DMSO Plates (serial 1:2 for9 points), Add 20 μl compounds at 10 mM in the 2nd column of 96 wellplate. Perform serial 1:2 across the plate (10 μl+20 μl 100% DMSO) for atotal of 9 points using Precision. Media Plates 96-well conical bottompolypropylene plates from Nunc (cat. #249946) (1:50 dilution) Add 147 μlof Media into all wells. Transfer 3 μl of DMSO+compound from each wellin the DMSO Plate to each corresponding well on Media Plate usingRapidplate.

Drug Addition to Cells, Cell Plate (1:10 dilution), Add 6 μl ofmedia+compound directly to cells (54 μl of media on the cells already).Incubate 3 days at 37 C, 5% CO2 in an incubator that will not be openedoften.

Day 5—Develop Plates, Thaw Cell Titer Glo Buffer at room temperature.Remove Cell Plates from 37° C. and equilibrate to room temperature. forabout 30 minutes. Add Cell Titer Glo Buffer to Cell Titer Glo Substrate(bottle to bottle). Add 30 μA Cell Titer Glo Reagent (Promega cat. #G7572) to each well of cells. Place on plate shaker for about 30minutes. Read luminescence on Analyst HT Plate Reader (half second perwell).

Cell viability assays and combination assays: Cells were seeded at1000-2000 cells/well in 384-well plates for 16 h. On day two, nineserial 1:2 compound dilutions are made in DMSO in a 96 well plate. Thecompounds are further diluted into growth media using a Rapidplate robot(Zymark Corp., Hopkinton, Mass.). The diluted compounds are then addedto quadruplicate wells in 384-well cell plates and incubated at 37° C.and 5% CO2. After 4 days, relative numbers of viable cells are measuredby luminescence using Cell-Titer Glo (Promega) according to themanufacturer's instructions and read on a Wallac Multilabel Reader(PerkinElmer, Foster City). EC50 values are calculated using Prism® 4.0software (GraphPad, San Diego). Formula I compounds and chemotherapeuticagents are added simultaneously or separated by 4 hours (one before theother) in all assays.

An additional exemplary in vitro cell proliferation assay includes thefollowing steps:

1. An aliquot of 100 μl of cell culture containing about 10⁴ cells inmedium is deposited in each well of a 384-well, opaque-walled plate.

2. Control wells are prepared containing medium and without cells.

3. The compound is added to the experimental wells and incubated for 3-5days.

4. The plates are equilibrated to room temperature for approximately 30minutes.

5. A volume of CellTiter-Glo Reagent equal to the volume of cell culturemedium present in each well is added.

6. The contents are mixed for 2 minutes on an orbital shaker to inducecell lysis.

7. The plate is incubated at room temperature for 10 minutes tostabilize the luminescence signal.

8. Luminescence is recorded and reported in graphs as RLU=relativeluminescence units.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. Accordingly, all suitablemodifications and equivalents may be considered to fall within the scopeof the invention as defined by the claims that follow. The disclosuresof all patent and scientific literature cited herein are expresslyincorporated in their entirety by reference.

We claim:
 1. A compound selected from Formula I:

or stereoisomers, tautomers, or pharmaceutically acceptable salts thereof, wherein: the solid/dash line

indicates a single or double bond; X¹ is CH or N; X² is CR² where R² is H or F; X³ is CH; Y¹ and Y² are independently selected from CH and N; Y³ is C or N; Y⁴ is CR⁶, N or NH; where one or two of Y¹, Y², Y³ and Y⁴ are N; R⁴ is selected from H, F, Cl, CN, and —CH₂OH; R⁵ is selected from —CH₃, —CH₂CH₃, —CH₂OH, —CH₂F, —CHF₂, —CF₃, —CN, and —CH₂CH₂OH; n is 0, 1, 2, 3, or 4; R⁶ is selected from H and —CH₃; R⁷ is selected from the structures:

where the wavy line indicates the site of attachment; R⁸ is selected from H, —CH₃, —S(O)₂CH₃, cyclopropyl, azetidin-3-yl, oxetan-3-yl, and morpholin-4-yl; and Z is CH.
 2. The compound of claim 1 having the structure of Formula Ia:


3. The compound of claim 2 having the structure of Formula Ib:


4. The compound of claim 1 selected from Formulas Ic-Ih having the structures:


5. The compound of claim 1 wherein X¹ is N, and R² if F.
 6. The compound of claim 1 wherein R⁴ is —CH₂OH.
 7. The compound of claim 4 wherein R² is F.
 8. The compound of claim 7 wherein X¹ and X³ are CH.
 9. The compound of claim 1 wherein Y⁴ is CR⁶, and R⁶ is CH₃.
 10. A compound selected from 2-(5-fluoro-2-(hydroxymethyl)-3-(8-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-a]pyridin-6-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one; 2-(5-fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridine-2-ylamino)imidazo[1,2-a]pyridin-6-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1 (2H)-one; 5-[5-fluoro-2-(hydroxymethyl)-3-{(8-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-a]pyridin-6-yl)}phenyl]-8-thia-5-azatricyclo-[7.4.0.0^(2,7)]trideca-1(9),2(7)-dien-6-one; 2-(5-fluoro-2-(hydroxymethyl)-3-(8-(5-(4-methylpiperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one; 2-(5-fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one; 2-(5-fluoro-2-(hydroxymethyl)-3-(1-methyl-5-(5-(4-(oxetan-3-yl)-1,4-diazepan-1-yl)pyridin-2-ylamino)-6-oxo-1,6-dihydropyridin-3-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one; 2-(5-fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2ylamino)imidazo[1,2-a]pyrazin-6-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one; 2-(5-fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-[1,2,4]triazolo[1,5-a]pyridin-6-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one; 2-(5-fluoro-2-(hydroxymethyl)-3-(3-methyl-7-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)-3H-benzo[d]imidazol-5-yl)phenyl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one; 10-[5-fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)phenyl]-4,4-dimethyl-1,10-diazatricyclo[6.4.0.0^(2,6)]dodeca-2(6),7-dien-9-one; 5-[5-fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)phenyl]-8-thia-5-azatricyclo-[7.4.0.0^(2,7)]trideca-1(9),2(7)-dien-6-one; 5-[5-fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)phenyl]-8-thia-4,5-diazatricyclo-[7.4.0.0^(2,7)]trideca-1(9),2(7),3-trien-6-one; 10-[5-fluoro-2-(hydroxymethyl)-3-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)phenyl]-4,4-dimethyl-7-thia-10-azatricyclo[6.4.0.0^(2,6)]dodeca-1(8),2(6)-dien-9-one; 2-(3-(hydroxymethyl)-4-(8-(5-(4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)pyridin-2-yl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one; and (S)-2-(3-(hydroxymethyl)-4-(8-(5-(2-methyl-4-(oxetan-3-yl)piperazin-1-yl)pyridin-2-ylamino)imidazo[1,2-b]pyridazin-6-yl)pyridin-2-yl)-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1(2H)-one.
 11. The compound of claim 1 selected from 2-[5-fluoro-2-(hydroxymethyl)-3-[7-[[5-[4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-3H-benzimidazol-5-yl]phenyl]-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1-one; 2-[3-(hydroxymethyl)-4-[7-[[5-[4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-3H-benzimidazol-5-yl]-2-pyridyl]-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1-one; 2-[3-(hydroxymethyl)-4-[8-[[5-[4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]imidazo[1,2-a]pyridin-6-yl]-2-pyridyl]-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1-one; and 2-[3-(hydroxymethyl)-4-[8-[[5-[4-(oxetan-3-yl)piperazin-1-yl]-2-pyridyl]amino]-[1,2,4]triazolo[1,5-a]pyridin-6-yl]-2-pyridyl]-3,4,6,7,8,9-hexahydropyrazino[1,2-a]indol-1-one.
 12. A pharmaceutical composition comprised of a compound of any one of claims 1-5 or 6-11 and a pharmaceutically acceptable carrier, glidant, diluent, or excipient.
 13. The pharmaceutical composition according to claim 12, further comprising an additional therapeutic agent.
 14. A process for making a pharmaceutical composition which comprises combining a compound of any one of claims 1-5 or 6-11 with a pharmaceutically acceptable carrier.
 15. A method of treating a disease or disorder which comprises administering a therapeutically effective amount of the pharmaceutical composition of claim 12 to a patient with an immune disorder selected from systemic and local inflammation, arthritis, rheumatoid arthritis, inflammation related to immune suppression, organ transplant rejection, allergies, ulcerative colitis, Crohn's disease, dermatitis, asthma, systemic lupus erythematosus, Sjogren's Syndrome, multiple sclerosis, scleroderma/systemic sclerosis, idiopathic thrombocytopenic purpura (ITP), anti-neutrophil cytoplasmic antibodies (ANCA) vasculitis, chronic obstructive pulmonary disease (COPD), and psoriasis.
 16. The method of claim 15 wherein the immune disorder is rheumatoid arthritis.
 17. The method of claim 15 further comprising administering an additional therapeutic agent selected from an anti-inflammatory agent, an immunomodulatory agent, chemotherapeutic agent, an apoptosis-enhancer, a neurotropic factor, an agent for treating cardiovascular disease, an agent for treating liver disease, an anti-viral agent, an agent for treating blood disorders, an agent for treating diabetes, and an agent for treating immunodeficiency disorders.
 18. A kit for treating a condition mediated by Bruton's tyrosine kinase, comprising: a) a first pharmaceutical composition of claim 12; and b) instructions for use. 